College of Agriculture

Department of Animal Sciences

Program: MSc in Animal Production

Course Title : Animal Biotechnology

By:
Aleme A (PhD, in Dairy Science and Technology)
 Chapter one: Introduction
What is Biotechnology?

Biotechnology: The development and utilization of biological

processes for obtaining maximum benefits to man and other forms of
life

Biotechnology: The use of living organisms for the manufacture of

useful products

 It may involve algae, bacteria, fungi, yeast cells of higher animal and
plants

Biotechnology – using living organisms, or the products of living

organisms, for human benefit to make a product or solve a problem
  Gene cloning
Examples
 Genetic engineering
– Fermentation
 Recombinant DNA technology
– Selective breeding
 Human Genome Project
– Use of antibiotics

Example of Biotechnology – Selective Breeding

A} B}

Normal zebrafish "Casper" zebrafish – made

by selective breeding
Biotechnology
 Provides new tools for improving human health and animal health and
welfare and increasing livestock productivity
 Improves the food we eat - meat, milk and eggs
 Improve an animal’s impact on the environment
 Enhances ability to detect, treat and prevent diseases
Just like other assisted reproduction techniques Such as
 Artificial insemination,
 Embryo transfer and in vitro fertilization,
 Livestock cloning improves animal breeding programs allowing farmers
and ranchers to produce healthier offspring, and therefore producer
healthier, safer and higher quality foods more consistently
 What is Animal Biotechnology and What Does It Mean to You?

Animal Biotechnology: is the application of scientific and engineering

principles to the processing or production of materials by animals to
provide goods and services
▪ The goal is to make products, to improve animals and to develop
microorganisms for specific agricultural uses
 Sometimes the animal biotechnology has been limited to genetic-
based biotechnology only
 However, the animal biotechnology uses the other, different
techniques, such as
 Artificial insemination
 Embryo transfer
 In vitro fertilization
 Embryo culture
 Cloning by nuclear transfer from embryonic or adult somatic cell
 Animals are playing a growing role in the advancement of
biotechnology, as well as increasingly benefiting from biotechnology

Combining animals and biotechnology results in advances in four

primary areas

1. Advances in human health

2. Improved animal health and welfare
3. Enhancements to animal products
4. Environmental and conservation benefits

 Animal biotechnology includes all animals: livestock, poultry, fish,

insects, and laboratory animals
Applications developed through research have led to the emergence of
three scientific agricultural animal biotechnology sectors:

1. Animal genomics

2. Animal cloning

3. Genetic engineering of animals

The Technology Sectors in the Animal Biotechnology Industry

Animal genomics: Genomics defines and characterizes the complete

genetic makeup of an animal

 By understanding the genomes of animals, we can better understand

the basis for disease resistance, disease susceptibility, weight gain,

and determinants of nutritional value

Animal cloning: Using somatic cell nuclear transfer, livestock
breeders can create an exact genetic copy of an existing animal –
essentially an identical twin

Cloning does not manipulate the animal’s genetic makeup nor

change an animal’s DNA: it is simply another form of sophisticated
assisted reproduction

Transgenic animals: A transgenic animal is one which has had

genetic material from another species added to its DNA

This breakthrough technology allows scientists to precisely transfer

beneficial genes from one species to another
 Animal Biotechnology to Advance Human Health

 Animals have been used for years to produce medicines for humans

 Animal-made pharmaceuticals (AMPs) transform biotech animals

into “factories” to produce therapeutic proteins in their milk, eggs,
and blood, which can be used in the development of
biopharmaceuticals

 In addition, biotechnology can be used to produce human-

compatible transplant organs, tissues and cells in pigs that can be
vital to enhancing human health
 Biotechnology to Improve Animal Health

For decades, farmers have been improving livestock herds through enhanced animal
husbandry practices and more modern technologies, such as

 Artificial insemination,

 Embryo transfer,

 In vitro fertilization,

 Genetic mapping and cloning

 Through biotechnology, farmers can enhance breeding, resulting in healthier herds

Additionally, the animal health industry has developed treatments that can prevent
and treat disease

 New vaccines, diagnostic tests and practices can help farmers treat animal
diseases, while reducing food borne pathogens at the farm level
 Biotechnology to Develop More Nutritious Food

• Improved animal health conditions from vaccines, medicines and

diagnostic tests result in safer foods for consumers

• In addition, food quality may be improved by introducing desirable

traits through new genes into farm livestock and poultry

• In the future, meat, milk and egg products from animals can be
nutritionally enriched with the use of biotechnology
 Conservation of Environment and Animals

• Biotechnology can help produce environmentally friendly animals,

as well as conserve endangered species

• Farm animals and their feeds have been improved through

biotechnology to reduce animal wastes, minimizing the impact on
the environment

• Today’s reproductive and cloning techniques offer the possibility of

preserving the genetics of endangered species

• Genetic studies of endangered animals can also result in increased

genetic diversity which can result in healthier populations of
species
 Origin and History of Biotechnology

• • Biotechnology:
What is the branch of molecular biology that studies the use of
microorganisms to perform specific industrial processes; "biotechnology

produced genetically altered bacteria that solved the problem"

Origins of “biotechnology” emerge in methods of food production and

plant and animal breeding

– Use of bacteria to produce cheese (food preservation)

– Use of natural enzymes in yogurt

– Use of yeast to produce bread

– Use of fermentation for producing wine and beer

 History of Biotechnology
Biotechnology in B.C.
500 BC: The Chinese use moldy curds as an antibiotic to treat boils
250 BC: The Greeks practice crop rotation to increase soil fertility
100 BC: Chinese use powdered chrysanthemum as an insecticide

Pre-20th Century Biotechnology

1590: Janssen invents the microscope

1663: Hooke discovers cells

1675: Leeuwenhoek discovers bacteria and protozoa

1797: Jenner inoculates a child with a viral vaccine to protect him from
smallpox

1802: 1st time the term “biology” is used

1830: Proteins, the building blocks of cells, are discovered

1833: The nucleus of the cell is discovered

1855: The E. coli bacterium is discovered

1855: Pasteur works with yeast, eventually proving they are living
organisms

1863: Mendel discovers genes while working with peas. He lays the
groundwork for genetics

1879: Flemming discovers chromatins

1883: The rabies vaccine is developed

1888: Waldyer discovers the chromosome

 Biotechnology in the first part of the 20th Century
1902: The term "immunology" first used
1906: The term "genetics" is used
1915: Bacterial viruses, called phages, are discovered
1919: The word "biotechnology" is first used
1927: Muller discovers that X-rays cause mutation
1928: Fleming discovers penicillin
1938: The term "molecular biology" is used
1941: The term "genetic engineering" is first used
1942: The electron microscope is used and characterizes viruses that infect bacteria,
called bacteriophages
1944: DNA is shown to be the building block of the gene
1949: Pauling proves that sickle cell anemia is a "molecular disease" caused by a
mutation
 Biotechnology in 1950s and 1960s

1953: Watson and Crick understand the structure of DNA

1954: Cell-culturing techniques are first used

1955: An enzyme involved in the production of a nucleic acid is

isolated

1956: The fermentation process is perfected

1960: Messenger RNA is discovered

1961: The genetic code is understood

 Biotechnology in the 1970s
1972: The DNA composition of humans is shown to be 99% similar to
that of chimps and gorillas

1977: Genetically-engineered bacteria are used to make human

growth protein

1978: North Carolina scientists, Hutchinson and Edgell, prove it is

possible to introduce specific mutations at specific sites in a DNA
molecule

1979: The first monoclonal antibodies are synthesized

 Biotechnology in the 1980s

1980: The U.S. Supreme Court approves the patenting of genetically-

engineered life forms

1980: The U.S. patent for gene cloning is awarded to Boyer and Cohen.

1981: The North Carolina Biotechnology Center is created—the 1st

state-sponsored research center for biotechnology

1981: The first genetically-engineered plant is reported

1981: 1st mice to be successfully cloned

1982: Humulin, human insulin drug, produced by genetically-

engineered bacteria (first biotech drug approved by the FDA)
1983: The first artificial chromosome is made
1983: The first genetic markers for specific inherited
diseases are found
1984: The DNA fingerprinting technique is developed
1984: The first genetically-engineered vaccine is developed
1986: The first biotech-derived interferon drugs for the
treatment of cancer are synthesized
1988: Congress funds the Human Genome Project
1989: Microorganisms are used to clean up the Exxon
Valdez oil spill
 Biotechnology in the 1990s

1990: The first federally-approved gene therapy treatment is

performed successfully

1992: The structure of HIV RT is elucidated

1993:The FDA declares that genetically engineered foods are "not

inherently dangerous"

1994: The first breast cancer gene is discovered

1996: Scientists clone identical lambs from early embryonic sheep

1998: Scientists clone three generations of mice from nuclei of
adult ovarian cells

1998: Embryonic stem cells are used to regenerate tissue and

create disorders that mimic diseases

1998: The Biotechnology Institute is founded by BIO as an

independent, national, 501(c)(3) education organization

1999: The genetic code of the human chromosome is

deciphered
 Biotechnology in the 2000 and beyond

2000: A rough draft of the human genome is completed

2000: Pigs are the next animal cloned by researchers to help
produce organs for human transplant
2001: The sequence of the human genome is published in
Science and Nature
2002: Scientists complete the sequence of the pathogen of rice,
a fungus that ruins enough rice to feed 60 million people
annually
2003: Dolly, the cloned sheep from 1997, is euthanized
 NEW (MODERN) AND OLD (TRADITIONAL) BIOTECHNOLOGY

Traditional (Old) Biotechnology

 Refers to the conventional technology which have been used for

many centuries. Beer, Wine, Cheese and many foods have been
produced using traditional biotechnology

 The Traditional biotechnology is an art rather than a science

Eg. Fermentation
 New (Modern) Biotechnology
 Capability of science to change the genetic material for genetic
new products for specific requirement through recombinant DNA
technology

Examples
– Gene cloning

– Genetic engineering

– Recombinant DNA technology

– Human Genome Project

 SCOPE OF BIOTECHNOLOGY
1. Health care:

(a) In 1982, human insulin (humulin) has been produced by

microorganisms in fermenters

(b) Hepatitis B vaccines (Recombivax HB), genetically engineered

vaccines produced biotechnologically
2. Gene Therapy:

 This is in a way, genetic engineering of humans, which would allow a person

suffering from a disabling genetic disorder to lead a normal life

3. Immunotechnologies:

 Monoclonal antibodies (MABs) for diagnosis and therapy

 Antibodies, special sets of proteins present in humans that enable them to fight
incursion of their bodies by harmful chemicals or microorganisms
Gene Therapy
 4. Tissue culture
 Tissue culture of both plant and animal cells

 These are used for Micropropagation of elite or exotic materials

(such as orchids), production of useful compounds such as taxol
(the widely used anti-cancer drug) and vanillin, and preparation in
the laboratory of “natural” tissues such as arteries for arterial graft
or skin for burn victims

5. Stem cell techniques:

 Which would involve purification and isolation of stem cells from

various tissues and develop into the desired tissue which could
then be used, for example, for transplantation
Stem cell techniques :
Stem Cell Therapy
 6. New DNA technologies:

 These include DNA fingerprinting, sequencing of genomes,

development and use of new molecular markers for plant
identification and characterization

7. Organotransplantation:
 Xenotransplantation that is transplantation into humans of organs
from other animal

 It appears that pig may be the most suitable for this biochemically,
anatomically and immunologically
 8. Bioremediation

• Bioremediation is the use of microorganisms to detoxify pollutants,

present in the environment usually as soil or water sediments
 9. Human Genome Project (HGP)
 Human genome has been sequenced and chromosome map has
been developed in various laboratories world-wide through
coordinated efforts

10. Bioinformatics
 Application of information sciences to increase the
understanding of biology, biochemistry and biological
data
 CHAPTER TWO : CELLS

The Basic Units of Life

 The Cell Theory

1. All organisms are made of cells

2. The cell is the basic unit of life in all living things

3. All cells come from existing cells

 THIS IS IMPORTANT BECAUSE IT SHOWS THAT ALL LIVING THINGS

SHARE A SIMILAR STRUCTURE
Types of Cells
 Two Types of Cells

Prokaryotic Cells:
• Have no membrane covered nucleus

• Have no membrane - covered organelles

• Have circular DNA

Eg. bacteria
Eukaryotic Cells:
• Have a nucleus

• Have a membrane -
covered organelles

• Have linear DNA

Eg. Plant and Animal cell

 Organelles

 Organelles are structures that enable the cell to live, grow and
reproduce

 Tell students that all cells have organelles?, however not all cells
have membrane covered organelles
 Cell Membrane

• The thin layer of protein and fat that surrounds the cell

• The cell membrane is semi permeable, allowing some substances

to pass into the cell and blocking others

Outer layer of cell

• Allows nutrients into the cell

and wastes outside of the cell

“Gate into the city”

 Cytoplasm

 Fluid filled interior of cell

Cytoplasm: a jelly-like fluid
contained in the cell that holds
the organelles/suspends cell
organelles
The Nucleus
 The Nucleus

-Surrounded by a nuclear envelope with nuclear pores

-Contains the genetic information (DNA) which exists as chromatin in
a non-dividing cell
-Controls all cell activities
-“Mayor’s office”

Nucleolus
-In the center of the nucleus
-Produces the Ribosomes
 Mitochondria

• Produces cellular energy in the form of ATP through a process called

Cellular respiration (metabolizes glucose)

• Power center of cell

• Provides the energy the cell needs to move, divide, etc.

“Electric company of the cell ”

 Ribosomes
 Site where proteins are made
 Site of protein synthesis
 Links amino acids together to form proteins
 Cell parts are made of proteins
“Factories of the cell”
 Endoplasmic Reticulum

 Transport system within the cell

 Transport cell materials

 “Roadways of the cell”

Two types

• Rough ER- ribosome's attached

• Smooth ER- no ribosome's

Rough ER
-Has ribosomes which give it its "rough" appearance
-Functions in protein synthesis
Smooth ER
-Mostly contains enzymes that function in lipid synthesis
 Golgi Apparatus

--Flattened stacks of membranes

--Collects, packages and modifies cell materials to be used in other parts
of the cell or transported out of the cell

Packaging house of cell

Packages, processes, and ships out the stuff the cell makes

“UPS of the cell”

 Lysosomes
-Digests and recycles old cell parts, sometimes bacteria
• -Contain high levels of enzymes

Digests food particles and cell parts

– “Garbage men”

• Protects cell by digesting foreign invaders

– “Police men ”
 Vacuole
-Mainly storage of foods, water and wastes
in plant cells, some waste
- Some animal cells have small vacuoles

• Vacuole

Vacuole is largest organelle in

plant cell
 Cell Wall

• Found only in plant cells

• Protects and supports the cell

 Chloroplasts

• Found only in plant cells

• Contains chlorophyll (makes plants green)

• Where photosynthesis takes place

 Centrioles

 Used during cell division to move and separate chromosomes

- Only found in animal cells
 Plant or Animal Cell?
Found in plant and Animal Cells

• Nucleus
• Golgi Complex
Found only in plant cell
• Mitochondrion •Chloroplasts
• Lyosomes
• Endoplasmic • Cell Wall
• Reticulum
• Cell Membrane
• Ribosomes
• Vacuoles
 CHAPTER THREE:
Molecular Biology and Genetic Engineering

• Molecular biology-The study of biology on a molecular level including the

structure, function, and makeup of biologically important molecules such
as DNA, RNA, and proteins
• Recombinant DNA technology-a set of techniques for manipulating DNA,
including: the identification and cloning of genes; the study of the
expression of cloned genes; and the production of large quantities of gene
product
• Genetic engineering-the process of transferring DNA from one organism
into another that results in a genetic modification
• Biotechnology-production of goods and services using biological
organisms, systems, and processes
• Molecular biotechnology-rDNA technology + biotechnology
Many scientific disciplines contribute to molecular biotechnology,
which generates a wide range of commercial products
 Molecular Biology Model Systems

• Viruses

• Bacteria (E. coli)

• Yeast (Saccharomyces cerevisiae)

• Round worms (Caenorhabditis elegans)

• Fruit fly (Drosophila melanogaster)

• Zebrafish (Danio rerio)

• Arabidopsis thaliana (thale cress)

• Mouse (Mus musculus)

Each experimental organism used in cell biology has advantages for
certain types of studies
 Central Dogma of Biology

Transcription Translation
• DNA RNA Protein $$$
Reverse transcription
Molecular
Biotechnology
DNA ---------→ RNA---------→Protein.

• This unidirectional flow equation represents the Central Dogma (fundamental

law) of molecular biology

• This is the mechanism whereby inherited information is used to create actual

objects, namely enzymes and structural proteins

• An exception to the central dogma is that certain viruses (retroviruses) make

DNA from RNA using the enzyme reverse transcriptase
 A Few Key Events Led to the Discovery of the Structure of DNA

 In 1953, James Watson and Francis Crick discovered the

double helical structure of DNA

 The scientific framework for their breakthrough was

provided by other scientists including
 Linus Pauling

 Rosalind Franklin and Maurice Wilkins

 Erwin Chargaff
 Linus Pauling

 In the early 1950s, he proposed

that regions of protein can fold
into a secondary structure
 a-helix

 To elucidate this structure, he built ball-

and-stick models
 Rosalind Franklin

 She worked in the same

laboratory as Maurice Wilkins

 She used X-ray diffraction to

study wet fibers of DNA
The diffraction pattern is interpreted
(using mathematical theory)

This can ultimately provide information

concerning the structure of the molecule
 Rosalind Franklin

 She made marked advances in X-ray diffraction techniques with

DNA

 The diffraction pattern she obtained suggested several structural

features of DNA

 Helical

 More than one strand

 10 base pairs per complete turn

 Erwin Chargaff’s Experiment

 Chargaff pioneered many of the biochemical techniques for the

isolation, purification and measurement of nucleic acids from living
cells

 It was already known then that DNA contained the four bases: A, G,
C and T

The Hypothesis

– An analysis of the base composition of DNA in different species

may reveal important features about the structure of DNA
 DNA -Composition and structure

• Two strands coiled called a double helix

• Sides made of a pentose sugar Deoxyribose bonded to phosphate

(PO4) groups by phosphodiester bonds

• Center made of nitrogen bases bonded together by weak hydrogen

bonds
DNA Double Helix

“Rungs of ladder”

Nitrogenous
Base (A,T,G or C)

“Legs of ladder”

Phosphate &
Sugar Backbone
 Helix

• Most DNA has a right-hand twist with 10 base pairs in a complete

turn

• Left twisted DNA is called Z-DNA or southpaw DNA

• Hot spots occur where right and left twisted DNA meet producing
mutations
 DNA

• Stands for Deoxyribonucleic acid

• Made up of subunits called nucleotides

• Nucleotide made of:

1. Phosphate group

2. 5-carbon sugar

3. Nitrogenous base
 DNA Nucleotide

Phosphate
Group

5
O=P-O CH2
O
O
N
Nitrogenous base
C4 C1 (A, G, C, or T)

Sugar
(deoxyribose)

C3 C2
 Pentose Sugar

• Carbons are numbered clockwise 1’ to 5’

5
CH2

C4 C1
Sugar
(deoxyribose)
C3 C2
 DNA
5 O 3

3 O
P 5 P
5 O
1 G C 3
2
4 4
2 1
3 5
P O
P
5
T A 3
O

O
5
P 3 P
 Antiparallel Strands

• One strand of DNA goes

from 5’ to 3’ (sugars)

• The other strand is

opposite in direction
going 3’ to 5’ (sugars)
 Nitrogenous Bases

• Double ring PURINES

Adenine (A)

Guanine (G) A or G

• Single ring PYRIMIDINES

Thymine (T)

Cytosine (C) T or C
 Base-Pairings

• Purines only pair with Pyrimidines

• Three hydrogen bonds required to bond Guanine & Cytosine

3 H-bonds

G C
•Two hydrogen bonds are required to bond Adenine & Thymine

T A
 DNA Replication
Replication Facts

• DNA has to be copied before a cell divides

• DNA is copied during the S or synthesis phase of

interphase

• New cells will need identical DNA strands

 Synthesis Phase (S phase)

• S phase during interphase of the cell cycle

• Nucleus of eukaryotes

S
phase
DNA replication takes
place in the S phase.
G1 interphase G2

Mitosis
-prophase
-metaphase
-anaphase
-telophase
 DNA Replication

• Begins at Origins of Replication

• Two strands open forming Replication Forks (Y-shaped

region)

• New strands grow at the forks

3’

Parental DNA Molecule

5’ Replication
Fork
3’

5’
 DNA Replication

• As the 2 DNA strands open at the origin, Replication Bubbles form

• Prokaryotes (bacteria) have a single bubble

• Eukaryotic chromosomes have MANY bubbles

Bubbles Bubbles
 DNA Replication

• Enzyme Helicase unwinds and separates the 2 DNA strands by

breaking the weak hydrogen bonds

• Single-Strand Binding Proteins attach and keep the 2 DNA

strands separated and untwisted

• Enzyme Topoisomerase attaches to the 2 forks of the bubble to

relieve stress on the DNA molecule as it separates
Enzyme Enzyme

DNA
 DNA Replication

• Before new DNA strands can form, there must be RNA primers
present to start the addition of new nucleotides

• Primase is the enzyme that synthesizes the RNA Primer

• DNA polymerase can then add the new nucleotides

 DNA Replication

• DNA polymerase can only add nucleotides to the 3’ end of the

DNA

• This causes the NEW strand to be built in a 5’ to 3’ direction

5’ 3’

5’
RNA
DNA Polymerase Primer
Nucleotide
 Remember HOW the Carbons Are Numbered!

Phosphate
Group

O
O 5
O=P-O CH2
O=P-O
O
O O
N
Nitrogenous base
C4 C1 (A, G, C, or T)

Sugar
(deoxyribose)

C3 C2
 Remember the Strands are Antiparallel
5 O 3

3 O
P 5 P
5 O
1 G C 3
2
4 4
2 1
3 5
P O
P
5
T A 3
O

O
5
P 3 P
 Synthesis of the New DNA Strands

• The Leading Strand is synthesized as a single strand from the

point of origin toward the opening replication fork

5’ 3’

5’

RNA
Nucleotides DNA Polymerase Primer
 Synthesis of the New DNA Strands
• The Lagging Strand is synthesized discontinuously
against overall direction of replication

• This strand is made in MANY short segments

• It is replicated from the replication fork toward the

origin

5’ Leading Strand 3’

3’ 5’
DNA Polymerase RNA Primer
5’ 3’

3’ 5’
Lagging Strand
 Lagging Strand Segments

• Okazaki Fragments - series of short segments on the lagging

strand

• Must be joined together by an enzyme

DNA
Polymerase

Okazaki Fragment
RNA
Primer
5’ 3’

3’ 5’
Lagging Strand
 Joining of Okazaki Fragments

• The enzyme Ligase joins the Okazaki fragments together

to make one strand

DNA ligase

5’ Okazaki Fragment 1 Okazaki Fragment 2

3’

3’ 5’
Lagging Strand
 Replication of Strands

Replication Point of Origin

Fork
 Proofreading New DNA

• DNA polymerase initially makes about 1 in 10,000 base

pairing errors

• Enzymes proofread and correct these mistakes

• The new error rate for DNA that has been proofread is 1
in 1 billion base pairing errors
 Semiconservative Model of Replication

• Idea presented by Watson & Crick

• The two strands of the parental molecule separate, and each acts
as a template for a new complementary strand

• New DNA consists of 1 PARENTAL (original) and 1 NEW strand of

DNA

DNA Template

Parental DNA
New DNA
 DNA Damage & Repair

• Chemicals & ultraviolet radiation damage the DNA in our

body cells

• Cells must continuously repair DAMAGED DNA

• Excision repair occurs when any of over 50 repair

enzymes remove damaged parts of DNA

• DNA polymerase and DNA ligase replace and bond the

new nucleotides together

copyright cmassengale 99
 Question:

• What would be the complementary DNA strand for the

following DNA sequence?

DNA 5’-CGTATG-3’
 Answer:

DNA 5’-CGTATG-3’
DNA 3’-GCATAC-5’
 RNA-Composition and structure

• It is formed of linear polynucleotide

• It is generally single stranded

• The pentose sugar is Ribose

• Uracil (U) replace Thymine (T) in the pyrimidine bases

Although RNA is generally single stranded, intra-molecular H-bond

base pairing occur between complementary bases on the same
molecule (secondary structure)
 The primary structure of an RNA strand is much like that
of a DNA strand

 RNA strands are typically several hundred to several

thousand nucleotides in length

 In RNA synthesis, only one of the two strands of DNA is

used as a template
  Although usually single-stranded, RNA molecules can form short
double-stranded regions

 This secondary structure is due to complementary base-

pairing

 A to U and C to G

 This allows short regions to form a double helix

 RNA double helices typically

 Are right-handed

 Have the A form with 11 to 12 base pairs per turn

 Different types of RNA secondary structures are possible

 Types of RNA
Messenger RNA (mRNA):Carries genetic information copied from DNA
in the form of a series of 3-base code, each of which specifies a
particular amino acid
Transfer RNA (tRNA):It is the key that read the code on the mRNA.
– Each amino acid has its own tRNA, which binds to it and carries it
to the growing end of a polypeptide chain
Ribosomal RNA (rRNA):Associated with a set of proteins to form the
ribosomes
– These complex structures, which physically move along the mRNA
molecule, catalyze the assembly of amino acids into protein chain
– They also bind tRNAs that have the specific amino acids according
to the code
 Types of RNA

1. Messenger RNA (mRNA) - Carries copies of instructions for the

assembly of amino acids into proteins from DNA to the rest of
the cell (serve as “messenger”)
2.Ribosomal RNA (rRNA) – Makes up the major part of ribosomes,
which is where proteins are made

Ribosomal
RNA
3. Transfer RNA (tRNA) - Transfers amino acids to ribosomes during
protein synthesis
 Proteins

• Proteins are made up of a chain of amino acids

• Proteins are enzymes, which catalyze and regulate chemical

reactions
 Steps to Make a Protein

1. Transcription
• DNA → RNA

2. Translation
• RNA → Protein (Chain of amino acids)
 When transcription needs to take place, DNA must provide the
code in order to create an mRNA strand.

 mRNA will be able to leave the nucleus and now it has the code
transcribed inside it’s base pairs!

 Practice:
DNA strand: TTA ACG GGT CTA
Matching DNA strand: AAT TGC CCA GAT
mRNA: UUA ACG GGU CUA
A segment of DNA has one strand with the following sequence of
bases: AGC GCA TAG CAA
The complimentary strand of RNA would be

a. UCG CGU AUC GUU

b. TCG CGT ATC GTT
c. AGC GCA UAG CAA
d. CTA TAC GCT ACC
 Gene Expression

• Genes are DNA sequences that encode proteins (the gene product)

• Gene expression refers to the process whereby the information

contained in genes begins to have effects in the cell

• DNA encodes and transmits the genetic information passed down

from parents to offspring
 Genetic code

• The alphabet of the genetic code contains only four letters

(A,T,G,C)

• A number of experiments confirmed that the genetic code is

written in 3-letter words, each of which codes for particular amino
acid

• A nucleic acid word (3 nucleotide letters) is referred to as a codon

Polymerase Chain Reaction(PCR)
 The Problem:
How do we identify and detect a specific sequence in a genome?

• TWO BIG ISSUES:

– There are a LOT of other sequences in a genome that we’re not interested in
detecting. (SPECIFICITY)

– The amount of DNA in samples we’re interested in is VERY small.

(AMPLIFICATION)
 How do we identify and detect a specific
The Problem: sequence in a genome?

Specificity

• Pine: 68 billion bp
• Corn: 5.0 billion bp
• Soybean: 1.1 billion bp
• Human: 3.4 billion bp
• Housefly: 900 million bp
• Rice: 400 million bp
• E. coli: 4.6 million bp
• HIV: 9.7 thousand bp
The Problem:

Specificity • How do we identify and detect a specific sequence in a genome?

• TWO BIG ISSUES:
Amplfication – There are a LOT of other sequences in a genome that we’re not
interested in detecting.
– The amount of DNA in samples we’re interested in is VERY small.

PCR solves BOTH of these issues!!!

PCR is an in vitro technique for the amplification of a region of DNA
which lies between two regions of known sequence
 Forensic DNA detection
So what’s PCR used for?
• Identifying transgenic plants

• Detection and quantification of

viral infection

• Cloning

• Detection of ancient DNA

• Gene expression analysis

 PCR History  In what has been called by some the greatest achievement

The Invention of modern molecular biology, Kary B. Mullis developed the

polymerase chain reaction (PCR) in 1983
 PCR allows the rapid synthesis of designated fragments of
DNA. Using the technique, over one billion copies can be
synthesized in a matter of hours
 PCR is valuable to scientists by assisting gene mapping, the
study of gene functions, cell identification, and to forensic
scientists in criminal identification
 Cetus Corporation, Mullis' employer at the time of his
discovery, was the first to commercialize the PCR process
 In 1991, Cetus sold the PCR patent to Hoffman-La Roche for
a price of $300 million
 It is currently an indispensable tool for molecular biologists
and the development of genetic engineering
 (1944 - )
The inventor of the DNA synthesis process known as the
Polymerase Chain Reaction (PCR). The process is an invaluable
Mr. PCR: Kary tool to today's molecular biologists and biotechnology
B. Mullis corporations.
Mullis, born in Lenoir, North Carolina, attended the University of
Georgia Tech for his undergraduate work in chemistry, and then
obtained a Ph. D. in biochemistry from Cal Berkeley.
In 1983, working for Cetus Corporation, Mullis developed the
Polymerase Chain Reaction, a technique for the rapid synthesis of
a DNA sequence. The simple process involved heating a vial
containing the DNA fragment to split the two strands of the DNA
molecule, adding oligonucleotide primers to bring about
reproduction, and finally using polymerase to replicate the DNA
strands. Each cycle doubles the amount of DNA, so multiple
cycles increase the amount of DNA exponentially, creating huge
numbers of copies of the DNA fragment.
Mullis left Cetus in 1986. For his development of PCR, he was co-
awarded the Nobel Prize in chemistry in 1993.
 The process, which Dr. Mullis conceptualized in 1983, is hailed as one of
the monumental scientific techniques of the twentieth century
A method of amplifying DNA, PCR multiplies a single, microscopic strand
of the genetic material billions of times within hours. Mullis explains:

The Invention of PCR
"It was a chemical procedure that would make the structures of the
molecules of our genes as easy to see as billboards in the desert
and as easy to manipulate as Tinkertoys....It would find infectious
diseases by detecting the genes of pathogens that were difficult or
impossible to culture....The field of molecular paleobiology would
blossom because of P.C.R. Its practitioners would inquire into the
specifics of evolution from the DNA in ancient specimens....And
when DNA was finally found on other planets, it would be P.C.R.
that would tell us whether we had been there before."

http://www.osumu.org/mu/events_lectures1b.htm
Practical Uses of PCR
  PCR’s ability to amplify even the smallest amount of
DNA from samples collected at a crime scene gives
the method great power when used in criminal
Uses of PCR: forensics
Forensics
 The DNA from body fluid, hair, or other tissue
samples is amplified to create a nearly unique
pattern for each individual
 This pattern can then be compared to suspects in
the case
 The infamous OJ Simpson case was the first one in
which the technique of PCR became widely
publicized
  Genetically-modified foods (GMOF)
are widely grown in the USA and
Uses of PCR:
GMO Food other countries
Detection  For various reasons, some
countries require exporters to
indicate the percentage of GMO
content in grain and food shipment
 PCR can be used to accurately
measure the exact quantity of
genetically-modified food in a
shipment, by “looking” at the DNA
that makes up the food!
  PCR’s power at identifying individual genetic
makeup has made it invaluable for use in
Uses of PCR: paternity testing
Paternity Testing
 By amplifying specific DNA fragments from
parents or close relatives, it is possible to
reconstruct relatedness between individuals
 PCR can not only identify relationships
between people today, but can also be used
to identify historical family relationships!
  PCR has been used for many scientific
studies in the field of archaeology:

Uses of PCR:
 Reconstructing the Dead Sea Scrolls
Archaeology

 Identification of paint pigments in cave

paintings

 Determining relatedness between individuals

in ancient ossuaries

 Constructing dinosaurs from blood preserved

in amber specimens (!)
  PCR is now invaluable in modern disease
Uses of PCR: diagnosis
Disease
Diagnosis  PCR can identify disease-causing organisms
much earlier than other methods, since it looks for
the DNA of the organism itself, not its proteins or
its effect on our immune system

 PCR has even been used to diagnose diseases of

the past, by amplifying minute amounts of
disease-related DNA in preserved specimens
Uses of PCR:
Disease  PCR can not only be used in disease
Treatment
diagnosis, but also as an aid in the
treatment of diseases

 For example, real-time PCR is used to

directly monitor the amount of HIV
virus in patients suffering from infection
 By monitoring the amount of virus
present, the drug therapy can be
continually adjusted to maximize virus
suppression
  Because PCR can be used to identify
not only individuals, but also can

Uses of PCR: differentiate between species, it is

Wildlife Conservation often used in wildlife conservation
research

 PCR can be used to monitor trade in

products made from endangered
species
 PCR can be used to monitor
ecosystems for the presence of certain
species
 PCR can be used even to monitor and
identify individual animals!
  The Human Genome Project has identified
tens of thousands of genes in the human
genome
Uses of PCR:  A key questions is: what do these genes
do? Part of the answer comes from
Basic Research determining when the genes are turned on
and off, and what affects the level of gene
expression
 Quantitative PCR is a key component of
determining the levels of gene expression,
and is a critical tool in cancer research,
disease studies, and developmental biology

DNA Enzymes
RNA

GENEX Analysis
Biology
The PCR Reaction Chemistry
 • Water
• Buffer
• DNA template
PCR Reaction • Primers
• Nucleotides
Components
• Mg++ ions
• DNA Polymerase
 • Water
PCR Reaction: – The medium for all other components
Water
 • Water

• Buffer
PCR Reaction:
Buffer – Stabilizes the DNA polymerase, DNA,
and nucleotides

– 500 mM KCl

– 100 mM Tris-HCl, pH 8.3

– Triton X-100 or Tween

 • Water
PCR Reaction: • Buffer
Template DNA
• DNA template

– Contains region to be amplified

– Any DNA desired

– Purity not required

– Should be free of polymerase inhibitors

 • Water
• Buffer
PCR Reaction:
• DNA template
Primers • Primers
– Specific for ends of amplified region
– Forward and Reverse
– Annealing temps should be known
• Depends on primer length, GC
content, etc.
– Length 15-30 nt
– Conc 0.1 – 1.0 uM (pMol/ul)
 • Water
PCR Reaction: • Buffer
• DNA template
Nucleotides • Primers
• Nucleotides
– Added to the growing chain
– Activated NTP’s
– dATP, dGTP, dCTP, dTTP
– Stored at 10mM, pH 7.0
– Add to 20-200 uM in assay
 • Water
• Buffer
• DNA template
PCR Reaction: • Primers
• Nucleotides
Magnesium
• Mg++ ions
– Essential co-factor of DNA polymerase
– Too little: Enzyme won’t work.
– Stabilizes the DNA double-helix
– Too much: DNA extra stable, non-specific
priming, band smearing
– Used at 0.5 to 3.5 uM in the assay
 • Water
• Buffer
PCR Reaction: • DNA template
Polymerase • Primers
• Nucleotides
• Mg++ ions

• DNA Polymerase
– The enzyme that
does the extension
– TAQ or similar
– Heat-stable
– Approx 1 U / rxn
Setting Up PCR Reactions
 Sterile Water 38.0 ul
10X PCR Buffer 5.0 ul
MgCl2 (50mM) 2.5 ul
dNTP’s (10mM each) 1.0 ul
PrimerFWD (25 pmol/ul) 1.0 ul
A Typical PCR PrimerREV 1.0 ul
DNA Polymerase 0.5 ul
Reaction DNA Template 1.0 ul

Total Volume 50.0 ul

 Component 1X 20X
Sterile Water 38.0 ul 760 ul
10X PCR Buffer 5.0 ul 100 ul
MgCl2 (50mM) 2.5 ul 50 ul
dNTP’s (10mM each) 1.0 ul 20 ul
Mixing Common PrimerFWD (25 pmol/ul) 1.0 ul 20 ul
Reagents Saves PrimerREV 1.0 ul 20 ul
DNA Polymerase 0.5 ul 10 ul
Time DNA Template 1.0 ul --

Total Volume 50.0 ul 980 ul

Aliquot
Add DNA
49 ul
as last step
An Even Simpler Sterile Water
10X PCR Buffer
Approach: MgCl2
Mastermix dNTP’s
DNA Polymerase
Primer FWD
Primer REV
DNA Template

MASTERMIX 19.6 ul
Sterile Water
10X PCR Buffer
MgCl2
dNTP’s
DNA Polymerase
Primers Fwd+Rev 0.4 ul
DNA Template 20.0 ul

Total Volume 40.0 ul

Programming the Thermal Cycler
 1. Initial Denaturation 95 C 3 min
Typical Thermal 2. DNA Denaturation 95 C 1 min
3. Primer Annealing 65 C 1 min
Cycler Conditions 4. Primer Extension 72 C 1 min
5. Go to step #2, repeat 39 more times
6. End
Analyzing the Amplified DNA
 •After thermal cycling, tubes are taken
out of the PCR machine.
•Contents of tubes are loaded onto an
PCR
Visualizing agarose gel.
Results •DNA is separated by size using an
electric field.
•DNA is then stained.
•PCR products are visible as different
“bands”.
 PCR
Visualizing
Results
 PCR
Visualizing
Results

Gel running
 After the gel has run, it is stained
to reveal the DNA bands:
PCR
Visualizing
Results
 The final result of the traditional
PCR procedure is a gel with a
PCR series of bands:
Visualizing
Results

Bands can be compared against

each other, and to known size-
standards, to determine the
presence or absence of a specific
amplification product.
 Genetic Engineering in Animals
Gene Transfer Technologies
 Definition
• It is defined simply as a technique to efficiently and stably

• introduce foreign genes into the genome of target cells

• The insertion of unrelated, therapeutic genetic information

• in the form of DNA into target cells

• Gene transfer methods majorly used in the treatment of

• diseases to supply patients with therapeutic genes

 Microinjection

▪ Microinjection is the process of transferring the desirable

DNA into the living cell ,through the use of glass
micropipette (0.5 - 5 micrometer)

• The micropipette easily penetrates into the cell membrane

and nuclear envelope

• The desired gene is then injected into the sub cellular

compartment and needle is removed
▪ Used to introduce DNA into large cells, normally performed
under a specialized optical microscope setup called
a micromanipulator.
 Advantages of microinjection:
▪ Frequency of stable integration of DNA is far better as
compare to other methods
▪ Method is effective in transforming primary cells as well
as cells in established cultures
▪ The DNA injected in this process is subjected to less
extensive modifications
▪ More precise integration of recombinant gene in limited
copy number can be obtained
Limitations of microinjection:

▪ Costly.

▪ Skilled personal required.

▪ Embryonic cells preferred for manipulation.

▪ Knowledge of mating timing, oocyte recovery is essential

▪ Method is useful for protoplasts and not for the walled

cells.
 Applications of microinjection
▪ Process is applicable for plant cell as well as animal cell
but more common for animal cells
▪ Technique is ideally useful for producing transgenic
animal quickly
▪ Procedure is important for gene transfer to embryonic
cells
▪ Applied to inject DNA into plant nuclei
 Electroporation

▪ Electroporation uses electrical pulse to produce transient

pores in the plasma membrane thereby allowing DNA into
the cells.

▪ These pores are known as electropores.

▪ The cells are placed in a solution containing DNA and

subjected to electrical pulse to cause holes in the membrane.

▪ The foreign DNA fragments enter through holes into the

cytoplasm and then to nucleus.
Advantages of
Electroporation

▪ Method is fast

▪ Less costly

▪ Applied for a number of cell

types

▪ Simultaneously a large
number of cell can be treated

▪ High percentage of stable

transformants can be produced
 General applications of electroporation

▪ Introduction of exogeneous DNA into animal cell lines,

plant protoplast, yeast protoplast and bacterial protoplast.

▪ Electroporation can be used to increase efficiency of

transformation or transfection of bacterial cells.
▪ Electroporation of early embryo may result in the
production of transgenic animals.
▪ Hepatocytes, epidermal cells, haematopoietic stem cells,
fibroblast, mouse T and B lymphocytes can be transformed
by this technique.

▪ Naked DNA may be used for gene therapy by applying

electroporation device on animal cells.
 Biolistics or Microprojectiles
▪ Biolistics or particle bombardment is a physical method
that uses accelerated microprojectiles to deliver DNA or
other molecules into intact tissues and cells.

▪ The gene gun is a device that literally fires DNA into

target cells .

▪ This method avoids the need of protoplast and is better in

efficiency.

 This technique can be used for any plant cells, root

section, embryos, seeds and pollen.

▪ The DNA to be transformed into the cells is coated onto

microscopic beads made of either gold or tungsten.
▪ The coated beads are then attached to the end of the plastic
bullet and loaded into the firing chamber of the gene gun.

▪ An explosive force fires the bullet with DNA coated beads

towards the target cells that lie just beyond the end of the
barrel.

▪ Some of the beads pass through the cell wall into the
cytoplasm of the target cells

▪ Here the bead and the DNA dissociate and the cells
become transformed.

▪ Once inside the target cells, the DNA is solubilized and

may be expressed.
Advantages and limitations of biolistics
Advantages

▪ Requirement of protoplast can be avoided.

▪ Walled intact cells can be penetrated.

▪ Manipulation of genome of sub-cellular organelles can be

achieved.

Limitations

▪ Integration is random.

▪ Requirement of equipments.
 Liposome mediated gene transfer

▪ Liposomes are spheres of lipids which can be used to

transport molecules into the cells
▪ These are artificial vesicles that can act as delivery
agents for exogenous materials including transgenes
▪ Promote transport after fusing with the cell membrane
▪ Cationic lipids are those having a positive charge are
used for the transfer of nucleic acid
 Advantages

▪ Simplicity.

▪ Long term stability.

▪ Low toxicity.

▪ Protection of nucleic acid

from degradation
 Calcium phosphate mediated DNA transfer

▪ The process of transfection involves the admixture of

isolated DNA (10-100ug) with solution of calcium
chloride and potassium phosphate so precipitate of calcium
phosphate to be formed
▪ Cells are then incubated with precipitated DNA either in
solution or in tissue culture dish
▪ A fraction of cells will take up the calcium phosphate DNA
precipitate by endocytosis
Transfection efficiencies is quite low
Application of gene transfer

▪ Clinical gene transfer applications

▪ Vaccine Development

▪ Production of transgenic animals

▪ Treatment of Cancer, AIDS

▪ Gene Discovery

▪ Gene Therapy

▪ GMO
 What are GM’s?

▪ Organism one that has been altered through recombinant

DNA technology

▪ Involves either the combining of DNA from different

genomes or the insertion of foreign DNA into a genome

▪ The most common genetically modified (GM) organisms

are crop plants

▪ Microbes are the first organisms to be genetically

modified
▪ GM technology is not a single methodology but a group of
methods which allow the manipulation of genes.

▪ Normally it involves the insertion of a functional gene

(plus its promoter, the equivalent of an on/off switch for
the gene, and a marker gene) into the genetic code of a
crop species

▪ Similar terms with GM

● Genetically engineered

● Transgenic

● Recombinant DNA (rDNA) technology

What is not a GMO?

❖GMO does not include;

❖ Mutants

❖ Fusion of animal cells unless the product can form an

animal.

❖ Plants formed x protoplast fusion.

❖ Plants formed by embryo rescue or in vitro fertilization

or zygote implantation.

❖ Organisms formed by natural DNA transfer.

The most common types of GMO‟S

FOODS

▪ Crops are modified to develop resistance to herbicides

and increase their nutrient content, for example corn
and soybeans.

▪ Fruits are modified to make them ripen later.

This help them available fresh in marketplace during a

longer time or for fruits that ripen after being picked,
make it easier to transport them.
The most common types of GMO’S

MEDICINES
▪These can be produced cheaper and easier some are:
insulin, thyroid hormones and the Hepatitis B vaccine

▪GM Bacteria‟s have been particularly important in

producing large amounts of pure human proteins for use in
medicine like clotting factors for hemophilia and human
growth hormones to treat dwarfism
 The most common types of GMO’S
MAMMALS
▪Research human diseases (To develop animal models for
many diseases.)

▪Produce industrial or consumer products (pharmaceutical

products or tissue implantation)

▪Enrich the animals‟ interactions with humans (Hypoallergic

pets)

▪Enhance production or food quality traits (faster growth

fish, pigs that digest food more efficiently)

▪Improve animal health (disease resistance)

 INSECTS
▪The effects of genetic changes on development (malaria
resistant mosquitoes).
AQUATIC LIFE
▪Evolution of immunity and developmental processes, rapid
growth (MADAKA -fish to detect pollutions in waterways)
 Spider silk
▪ Spider‟s silk is 5 times
stronger than a thread of steel of
the same thickness 3 times
stronger than Kevlar (carbon
fibre)

▪ Spiders are carnivores and

cannot be raised

▪ Transgenic animals can

produce the spider protein
▪ The gene for spider silk protein
was isolated from the golden orb
weaver (Nephila clavipes)

▪ Not easy, it is a fibrous protein

so it has a very repetitive gene
sequence
Goat that produce spider silk
▪ Two key genes that allow a spider to weave
their silk inserted into their genetic code.

▪ Gene (plus regulator genes) inserted into goat mammary

gland cells
▪ Genetically transformed cells could be made to secrete
spider silk protein
▪ Transformed goat cells fused to enucleated oocyte
▪ Genetically transformed goat embryos produced
▪ Goat grows up, females produce spider silk protein in their
milk
▪ Silk protein purified
▪ Extruded through nozzle to produce silken thread
▪ Not easy as the conditions are not quite the same a spider‟s
spinneret
▪ Transformed goats can be bred together perpetuating the
trait
  Goat that produce spider silk
▪ Stronger than steel and more flexible

▪ Used to replace damaged tendons and ligaments , suture

damaged eyes, or even nerves

▪ Make stronger and safer parachutes for soldiers ,

bulletproof vests
▪ Goats are milked
▪ Milk is frozen and the cream is separated
▪ Thawed milk is pushed into a micro filter that blocks the
larger fat molecules and lets the smaller proteins through

▪ A smaller filter then further isolates the silk proteins

▪ When dried looks like a white powder
 Applications of Spider silk

▪ Super strong surgery thread

▪ Artificial ligaments

▪ Light bullet proof body armour

▪ Biodegradable fishing line

 Advantage of GMO

▪ Animals can be engineered to require less food, grow

quicker, and leave behind less environmentally damaging
waste
▪ Animals can be engineered to be more resistant to harmful
and painful diseases
▪ Animals can be engineered to produce more omega-3 fatty
acids, to provide leaner meat, and to make more milk

▪ Animals can be engineered so their tissues, organs, and

cells can be transplanted into humans
▪ Animals can be engineered to produce certain substances
that offer a new source of medicine
▪ Animals can be engineered to reproduce much faster
 Disadvantage of GMO

▪ It is unethical
▪ Some food companies have refused to use meat or milk
that is from genetically engineered animals
▪ Some consumers are complaining that the animal drug
rules do not regulate GM animals properly
▪ This process is potentially dangerous and can be very
harmful
▪ When engineering animals the natural ecosystem can be
disturbed
▪ Some animals die in experiments while other are born
deformed or huge
▪ Animals may live in odd conditions that are affect their
natural way of life
What are the ethical issues? On GMO
Is it ethical?
 What is a transgenic animal?

▪ Transgenic animal: An organism that has had part of

another species' genome transferred into its own through the
techniques of genetic engineering

• An animal contains a foreign gene (genes) introduced

purposely by human intervention

• Transgenic animals are altered so that their DNA produce

proteins that normally they would not produce
 History of Transgenic Animals

▪ 1970's, first transgenic mice via viral infection, but not

germ-line transmission

▪ 1980's, first transgenic mice via microinjection, the most

popular technique

▪ 1985, first transgenic rabbits, sheep, pigs and cattle

▪ 1990's, transgenic farm animal companies as bioreactors

and organ donors
Different ways to create transgenic animals
❖ DNA Microinjection
❖ A foreign gene is directly injected into a fertilized egg
that is put into a female animal that acts as a surrogate
mother for the egg
❖ Retrovirus-Mediated Gene Transfer
❖ A retrovirus is a virus that attaches to an organism‟s
DNA and changes it to include a new characteristic.
Scientists expose ordinary cells to a retrovirus when they
are trying to create transgenic animals
❖ Embryonic Stem Cell-Mediated Gene Transfer
❖ Stem cells are blank cells that can turn into any type of
cell.
Scientists modify these cells, and then add them to an
embryo, which is a fertilized egg that develops and grows
 DNA Microinjection

▪ Direct microinjection of a chosen gene construct from

another member of the same species or from a different
species, into the pronucleus of a fertilized ovum.
Retrovirus mediated gene
transfer
▪ Retroviruses are used as
vectors
▪ Infects host cells
▪ Not all cells carry the
retrovirus
▪ The retrovirus must
integrate into the germ cells
to work.
Embryonic stem cell-mediated gene transfer
• Genetically engineered
embryonic stem (ES) cells can
be used to create transgenic
animals

• This method allow for gene

targeting via homologous
recombination.
▪ Insertion of desired DNA sequence into an in vitro
culture of embryonic stem (ES) cells.

▪ Stem cells can give rise to a complete organism.

▪ The cells are then incorporated into an embryo at the

blastocyst stage of development.
 Classification of Transgenic Animals

Five major categories:

1. Disease models: (e.g onco-mouse, AIDS mouse etc)

2. Transpharmers: (producing milk, drugs, proteins)

3. Xenoplanters: not express the foreign antigens

4. Food sources: (e.g super pig, super fish)

5. Scientific models: (e.g ANDi first transgenic monkey)

 Pros and Cons

▪ Pros
• Cheap
• Short generation time
• Defined genes
▪ Cons
• Ethical Concerns
▪ Are the animals treated with respect?
• Environmental Issues
▪ What if a genetically modified animal
gets loose?
• Religious Views
▪ Do we have the right to act as God?
FIGURE 22–9 Transgenic Atlantic salmon (bottom) overexpressing a
growth hormone (GH) gene display rapidly accelerated rates of
growth compared to wild strains and nontransgenic domestic strains
(top). GH salmon weigh an average of nearly 10 times more than
nontransgenic strains
FIGURE 22–10 Transgenic cows for battling mastitis. The mammary glands of nontransgenic
cows are highly susceptible to infection by the skin microbe Staphylococcus aureus.
Transgenic cows express the lysostaphin transgene in milk, where it can kill S. aureus before
they can multiply in sufficient numbers to cause inflammation and damage mammary tissue.
FIGURE 22–11 GloFish, marketed as the world’s first GM-pet
Applications of transgenic farm animals

The Rationale for creation of Transgenic Animals includes:

• Improving the commercial value of farm animals and

Pharming

• The use of farm animals as bioreactors that produce

pharmaceuticals or organs for xenotransplant.

• Basic research/disease model

 CHAPTER FOUR:
CLONING (ASEXUAL REPRODUCTION)
 What is Cloning ?

▪ Cloning is the creation of an organism that is an exact

genetic copy of another

 It is used to amplify DNA fragments containing whole

genes

▪ It is a process in which we produce a „genetic‟ twin of

living thing, with the organisms that starts life with the same
genes as its parents
Cloning: a very brief history
–1997: Dolly--first successful cloning of adult animal
–1997: Rhesus monkeys cloned
–1998: Cloned calves reported
–2001: Cloned pigs reported
–2001: Cloned gaur--first endangered species
–2001: First reported human embryo cloning
–2002: First cloned pet (cat)

•
• ?
 Why we want to do cloning

 Researchers hope that these techniques can be used in

researching and treating human diseases and genetically
altering animals for the production of human transplant
organs.

 We need to study human cloning to know if it is good or

not, or to know if it can help us or not
 Steps in Cloning
Step 1: Take any cell from your body, from the skin, for
example.
Step 2: Take an egg cell (ovum), from the ovary of any
woman.
Step 3: Take the nucleus out of the egg cell.
Step 4: Put together the cell of your skin and the egg
without nucleus. It will start to multiply forming a
microscopic ball of many identical cells.
Step 5: In about 6 days place it in the uterus of the woman.
Step 6: In 9 months a baby will be born just like you, an
identical twin of you… without any genetic
characteristics of the woman who gave the ovule
and provided the uterus, and gave birth to your twin.
 Methods of cloning
▪ Embryo splitting: Artificially splitting a single embryo at
a very early stage of development.

In the natural process this would create twins.

However, because this is done at an early stage and there are

usually less than eight cells you can only make a few clones.

Both the nuclear genes and mitochondria genes would be

identical.
▪Nuclear replacement: Genetic material (nucleus from
embryonic, fetal, or adult cell) is removed and placed into
an unfertilized egg or embryo, whose nucleus has been
removed.
In this case the nuclear genes remain the same but the
mitochondria DNA would be different.

This has the potential to create the clone of an adult

organism as well as many clones at once.

The three types of Cloning

1- DNA cloning/ Gene cloning

2- Reproductive cloning (Dolly)

3- Therapeutic Cloning
DNA cloning/ Gene cloning
▪ It is also called Recombinant DNA Technology or
"molecular cloning”
▪ Refers the transfer of a DNA fragment of interest from one
organism to a self-replicating genetic element such as a
bacterial plasmid.
Cloning strategies:
• Fragmentation: breaking apart a strand of DNA
• Ligation: gluing together pieces of DNA in a desired
sequence
• Transfection: inserting the newly formed pieces of DNA
into cells
• screening/selection: selecting out the cells that were
successfully transfected with the new DNA
▪ Used for large protein production, virus study and vaccine
production
Reproductive cloning
▪ It is used to generate an animal that has the same nuclear
DNA as another currently or previously existing animal.

▪ Dolly was created by reproductive cloning technology in a

process called "somatic cell nuclear transfer" (SCNT)

▪ In SCNT scientists transfer genetic material from the

nucleus of a donor adult cell to an egg whose nucleus, and
thus its genetic material, has been removed.

▪ If the egg begins to divide normally it is transferred into the

uterus of the surrogate mother.

▪ Such clones are not strictly identical since the somatic cells
may contain mutations in their nuclear DNA.
Generate a “twin animal”: same DNA (Dolly)
Dolly was the first mammal to have been successfully
cloned from an adult cell.
The entire animal is produced from a single cell by asexual
reproduction. This would allow for the creation of a human
being who is genetically identical to another.
Dolly in detail
▪ Dolly was cloned using the nuclear replacement method.
Again the nucleus with chromosome sets is fused with an
unfertilized egg whose nucleus has been removed.

▪ Motivating factor was that it could help to improve certain

qualities in livestock.
▪ Dolly was not the first sheep to be created from nuclear
replacement.

Two genetically identical sheep, Megan and Morag were

born in 1996 using the technique.

The difference was that Dolly was derived from an adult

sheep, and Megan and Morag were from a sheep embryo.
 Therapeutic Cloning
▪ Also called "embryo cloning," is the production of human
embryos for use in research.
▪ The goal of this process is not to create cloned human
beings, but rather to harvest stem cells that can be used to
study human development and to treat disease.
▪ “Production of human embryos for use in research: Stem
cell research”
 Other types of cloning

▪ Multiple copies of genes or gene fragments, repeating

nucleotide sequences

▪ Single cell organisms, like bacteria and fungi.

This includes fermentation processes for production of

bread, beer, and wine.

▪ Entire plant asexual replication

▪ Natural cloning occurs in sexual reproduction, when the

embryo splits in two to produce twins.
 Pros and Cons of Cloning
Pros:
• Produce animals with desirable traits.
• Increase the efficiency of the livestock production.
• Offset losses of among endangered species populations.
• Enable better research for finding cures to many diseases.
• Provide children for parents who would like a child but
can't have one for various reasons.
• Provide parents with an opportunity to clone a child who
has died.
Cons:
• Decline in genetic diversity.
• Taking nature into our own hands.
• Religious and moral reasons.
• Physical problems, such as birth defects.
• Possibility of mental and emotional problems of the clone.
Applications of Animal Cloning
1. Biomedical research
• Animals as drug producers
• Animal models
• Breeding androgenic body tissue
• Xenotransplantation
2. Livestock breeding and agriculture
• Transgenic clones
• Changes to agricultural structures

Dolly with her foster

mother

• Mitochondria???
Factors Affecting Organ Transplantation
▪ Longevity (life span)
▪ Organ size difference
▪ Hormone and Protein differences
▪ Environment the organ adapted to
Phases of Xenotransplantation Rejection
• Hyper Acute Xenograft rejection (HXR)
• Acute vascular xenograft rejection (AcXR)
• Cellular xenograft rejection
• Chronic xenograft rejection
 Methodology

▪ What three methods are scientists using to make

transplanted tissue unrecognizable to the immune system?

1. Suppressing the immune system.

2. Genetically engineering the donor cells to produce

human proteins that disguise the cells so the immune
system thinks they are part of their own tissue.

3. Giving the donor cells a seaweed-based „invisibility‟

coat.
Solutions to the Problems

▪ Selective breeding
▪ Genetic alterations
▪ Containment
▪ New and more powerful drugs
▪ More Research
 CHAPTER 5:
REPRODUCTIVE TECHNOLOGIES

Artificial insemination

Why is reproduction important? What is the process of animal reproduction?

• Reproduction is the process by which animals produce

offspring.

• The ability to reproduce is one of the basic characteristics

of a living thing; in order for a species to continue they
must be able to produce viable offspring.
 What Is AI?

• General definition-the introduction of semen into the oviduct or

uterus by some means other than sexual intercourse

• The use of semen from a genetically superior male to inseminate a

female resulting in a genetically superior offspring
 Where Is AI Used?

• Cattle • Turkey
• Horses • Chickens
• Swine • Rabbits
• Sheep • Fish
• Goats • Fox
• Dogs • Mink
• Humans • Bees
 Why Use AI?

• AI provides the producer an opportunity to use sires

possessing superior genetics
• Depending upon the needs and goals of an individual's
breeding program, AI offers an economically feasible
means of increasing productivity over a wide range of
traits
• In spite of this enormous potential to improve production
levels, many producers have not put themselves in a
position to take advantage of the benefits offered by AI
 Advantages

• Best possible sires of proven quality

• Disease control

• Cost effectiveness

• Flexibility

• Safety

• No bull, stallion, ram, boar, etc.

 Fertility Testing

• Important step in evaluating males to be kept for

breeding

• Indispensable in the area of artificial insemination since a

bull may be worth thousands of dollars & still produce
progeny long after his death (because the semen is
stored)

• The process of evaluating the bull's ability to reproduce.

 Factors to consider in Testing

• Libido (sex drive) - This is especially important in bulls kept for

natural breeding since some bulls are more active breeders than
others

• Scrotal circumference - This is an observable, measurable

characteristic that indicates fertility

• In general, the greater the scrotal circumference, the greater the

sperm production, hence increased fertility

• Semen evaluation- This is the most important factor in fertility

testing. Semen is evaluated for:
 Semen Evaluated For Appearance

• A uniform solid, dense appearance indicates a high sperm

concentration

• A sample which appears to be translucent contains fewer sperm

cells

• Semen with a curd appearance should not be used as this indicates

inflammation
 Semen Evaluated For Volume

• May vary depending on the age of the bull or sire

• Younger sires produce a smaller volume of semen which is not a

negative factor as long as the concentration of semen is good

Motility

• It must be evaluated under a microscope at 400X

• Most semen should contain 70 percent or more motile cells

 Concentration

• Determined by the number of sperm per milliliter of semen

• This is probably the most important indicator of fertility, except

with sires, in which scrotal circumference is the determinant

Morphology

• The proportion of abnormal sperm should not exceed 20 percent

• Most males do, however, produce some abnormal sperm

 Semen Collection Methods:
Artificial Vagina in goats and cattles

• Consists of an outer tube or casing which is constructed of heavy

rubber or plastic, and an inner tube or lining of thin rubber.

• The space between the two tubes is filled with warm water, which
maintains the collected sperm at a constant temperature, neither
too hot or too cold

• One end of the apparatus is open to allow the entrance of the

penis and the other end is attached to a glass tube or beaker to
receive the ejaculated semen.

• This is the most commonly used device for collecting semen

 Semen Collection Methods:
Electric Stimulation

• An electro-ejaculator apparatuses introduces a weak alternating

current to the sacral and pelvic nerves via electrodes placed in the
rectum until erection and ejaculation occur

• It is used on all farm animals except boars and horses

• It is used effectively on bulls that cannot mount due to leg injuries

 Semen Preparation: Storage & Shipment

• It may be kept at room temperature if it is to be used within two

hours of collection

• If the semen will be put into long-term storage, semen needs to be

gradually cooled and frozen a temperature below zero
 Semen Preparation: Semen Extenders
• Why Added?
• Add extra needed Volume
• Exert beneficial effect on the sperm

1. Egg yolk – phosphate: used in bull, ram, and stallion semen

2. Egg yolk – citrate: used in bull and ram semen

3. Homogenized whole milk: used in bull, ram, and boar semen
4. Glycine-containing diluents: used mostly in boar semen
5. Some extenders contain antibiotics to control bacteria and
contamination
 Equipment Needed For A.I.
CATTLE, GOATS AND HORSES

1. Liquid nitrogen tank

– Used to store semen straws at -320 degrees Fahrenheit

– Semen stored in tank, may last indefinitely once froze properly &
temperature is maintained
 Equipment Needed For A.I. cont.

2. A semen straw

– containing a single dose of semen

3. Straw tweezers

– Used to pick up the semen when in the tank & thawing unit
 Equipment Needed cont.
4. Semen Thawing Unit
– Used for thawing/softening the frozen semen before
insemination

– Contains water

5. Thermometer

– To measure the temperature that is in the thawing unit

– Temperature should be between 95 – 98 degrees Fahrenheit

 Equipment Needed cont.

6. Inseminating syringe or gun

– Made from stainless steel

– Used to place semen in the reproductive tract of the cow or

heifer and to maneuver it through the cervix

7. Sterile Lubricant (Lube)

– Lubrication put on plastic sleeve in order to first enter the

vulva
 Equipment Needed cont.

8. Paper Towels

– To dry off the semen straw

– To wipe animal clean, especially the vulva

– To put on top of the vulva to keep clean

9. Scissors

– To cut the end of the straw just prior to insemination

 Equipment Needed cont.

10. Plastic Sleeve

– To put on inseminator’s arm to keep clean

11. Cover Sheath

– Inseminator’s gun is put inside of the sheath so that all is kept

clean
 Equipment Needed cont.

12. KAMAR heat strips:

 Heating aid with red dye that expels when a cow or heifer is
mounted

13. Tail Chalk:

 Chalk paint is smeared when cow

or heifer is mounted
 Steps to AI
(Semen Storage)

• Collected, evaluated, cooled slowly, & frozen

– -320°F
• Lasts several months

• 30 years

• 40 years
 Steps to AI
(Heat Detection)

• Increased activity

• Mounting

• Swelling and redness of vulva

• Discharge

• Winking

– Mares

• Standing heat

– Best indicator of estrus

 Steps to AI (Insemination)
• Cattle
– 12 hours after heat detection
• Horses
– 3rd, 5th, & 7th day of estrus Horses
• Swine
– 24 and 12 hours after onset of estrus in sows
– 12 and 24 hours after onset of estrus in gilts
• Sheep
– To inseminations increase conception and multiple lambs
 Recto-vaginal Method (Cattle)

• Remove all feces from the rectum

• Grasp the cervix through the wall of the rectum

– Not done with horses or swine

• Insert inseminating tube through vagina and into the cervix

• Guide tube through cervix

• Deposit semen from middle of cervix to body of uterus

Recto-vaginal Method
 Lapriscopic Artificial Insemination (Sheep)

Semen injected from a syringe through a pipette into upper one-third

of uterine horn through abdominal wall
 Artificial Insemination procedures

Step #1: Restrain the animal to be inseminated

Step #2: Raise the tail with the right hand and gently massage the
rectum with the lubricated glove on the left hand

Step #3: Gently wipe the vulva with a paper towel to remove excess
manure and debris

Step #4: Insert the gun at a 30° upward angle to avoid entering the
urethral opening and bladder located on the floor of the vagina
Procedure for artificial insemination

Figure #1: Keeping the gloved hand

even with the tip of the inseminator gun

Figure #2: Allowing manure to pass

over the top of the hand and arm
 Procedure for artificial insemination

Figure #3: Dealing with colon constrictions

Figure #4: Grasping the cervix and gently moving it forward

Procedure for artificial insemination
Figure #5: Close-up of the cervix.

Figure #6: Finding the opening of the cervix.

 Procedure for artificial insemination
Figure #7: Moving the cervix over the tip of the insemination gun.

Figure #8: Locating the end of the insemination gun

 Procedure for artificial insemination
Figure #9: Depositing the semen in the body of the uterus

Figure #10: Good distribution of the semen to both uterine horns

 Procedure for artificial insemination

Figure #11: Improper distribution of the semen into one horn because
the insemination gun is pushed too far forward
 Advantages of AI
• Genetic Improvement

– Wide spread use and availability of genetically superior sires

– 1 bull can breed 500,000 cows in a lifetime

– After death, semen can be used

• Oldest frozen semen 40 - 45 years old

• Rapid proof of sire

– Progeny testing examines offspring for desired traits

– With natural mating would only have 100’s of offspring

 Advantages of AI (cont.)

• Availability of sires
– Sires anywhere in world

• Danger of bull (male) removed

• Disease reduction
• Crossbreeding
– Can try without buying sire

• Improved management
– Start to keep records
 Advantages of AI (cont.)

• Economics
– Cost of very good sire is reduced because extend
semen

– Cost to maintain sire’s reduced as don’t need as many

to breed all the females
 Disadvantages

• Estrus detection must be good

• Trained inseminator

• Bull semen the best, other species not as good

• Use of poor male may increase if not tested well

• Technology to store cooled or frozen semen

– Difficult to maintain
 Age When Semen Can Be Collected

Bull 12 months
Boar 6 - 8 months
Ram 6 - 9 months
Stallion 20 - 24 months
Dog 8 - 12 months
 Preservation of Semen

• Extenders (7 components)
– Nutrients
• Glucose, fructose

– Cold shock prevention

• Milk, skim-milk, egg yolk

– Buffer
• Citrate, Tris

– Osmotic pressure
• The buffer component
 Preservation of Semen (cont.)
– Inhibit bacterial growth
• antibiotics
– Increase volume
– Cryoprotectant
• Glycerol
• Liquid Semen
– Collect semen
– Semen quality exam
– Extend 1:3 (semen:extender)
• Minimal extension rate
– Cool to 5°C over 2 hours
• OK for bull, stallion, ram
• Boar - cool to 15°C
 Preservation of Semen (cont.)

– Once cooled, extend semen to final amount

• Bovine (inseminate 0.5 ml)

– 2 to 5 million sperm/ml

• Equine (inseminate 1 billion sperm)

– 25 to 50 million sperm/ml

– If don’t cool then inseminate 500 million motile sperm

• Swine (inseminate 1.5 to 6 billion sperm in 50 ml)

– 30 to 120 million sperm/ml

 Preservation of Semen (cont.)

• Frozen semen
– Follow instruction for collecting and cooling semen
– After cooling to 5°C, extend to 2X the final
concentration desired
• If want final concentration to be 40 million/ml then dilute to
80 million to ml at this time

– Hold semen for 4 to 6 hours at 5°C

• Equilibrates semen to the cold
 Preservation of Semen (cont.)

– Add the cryoprotectant

• Mix extender with 2X final cryoprotectant amount, 1:1 with
extended semen
• Do this in small portions to minimize cryoprotectant toxicity
– Package semen
• 0.5 ml French straws
• Ampules
– Freeze semen
• Liquid nitrogen vapor
– Static
– Mechanically controlled
• Dry ice depressions for pellet freezing
 Use and Success of AI

Semen
Species Liquid Frozen Preg. Rate Major Problems

Dairy Cattle OK OK 60-70 OK, need good heat

detection

Beef Cattle OK OK 55-65 Range area large:

poor heat detection
Sheep OK Fair 50-65 Large range; low value
of ewe
Swine OK Fair 40-75 Estrus detection
Horses OK Fair 30-60 Timing insemination,
breed restrictions
Turkey OK Poor 0-90 None
Humans OK Fair 5-30 Donors; infertility; time
Semen Sexing
 Semen Sexing

 Is the determination of sex based on sex chromosomes

 Sex is determined in many ways

1. Based on incubation temperature e.g.. Reptiles

 Certain reptiles become male or female depending on the

temperature that the eggs are incubated

 If the eggs of alligators are incubated below 30 oC, they become

females

 If incubated above 34 oC, they become males

2. Determined by sex chromosomes
 Sex chromosomes are chromosomes that differ in the two sexes
 One sex is homogametic or has a pair of the same sex
chromosomes
 The other sex is heterogametic or has two different chromosomes
 Examples of species with homogametic males and heterogametic
females are butterflies and birds
 Male = ZZ
 Female = ZW
 If the animal has a Y chromosome, it is a male;
 If it lacks a Y, it is a female
• Semen contains roughly 50% each of male and female sperm

• Sperm identical in size, weight, swimming speed etc.

METHODS OF SEMEN SEXING

 Fluorescence Activated Cell Sorting

 Use DNA Technology

 Fluorescence Activated Cell Sorting

• Sperm stained with a harmless, DNA‐binding dye (Hoecsht 33342)

• Sperm injected into flow cytometer in single file at 60 mph, 40 psi.

• Crystal vibrator breaks stream into individual droplets

• Sperm pass in front of a laser beam, stained DNA emits fluorescence

• Fluorescence is measured and a +ve or –ve charge is applied to each

single droplet

• Sperm pass between charged deflector plates: +ve droplets go one

direction, ‐ve another

• Uncharged droplets pass through as waste

 Limitations of FACS

 Some sperm cannot be oriented for sorting

 Low sorting speed
 Sperm damage caused by sorting process
 Some sperm cannot accurately be identified as bearing X or Y
chromosome
 Combined loss = 75% of original semen sample
 Only 10‐15% of original sample is marketable, sexed semen
 Reduced conception rates in sexed vs conventional semen
 Recommended for use in heifers only
 Cost = ~twice the price of conventional
 Excellent straw management and handling required
 Sexed semen use

• Published studies comparing CR with frozen semen

• Frozen sexed ~80 % of conventional
 I'e. conventional CR = 50%, Sexed CR = 40%
 ie. conventional CR = 70%, Sexed CR = 56%
Fresh sexed semen
 Avoids losses associated with freeze‐thaw process (approx 50%)
 Collect, sort and inseminate within 24‐36 hrs
 Well suited to seasonal systems
 Potential to extend sexed semen use to all cows
 Main problem = availability during peak demand
 Benefits of sexed semen use

 90 % heifer calves
 Greater numbers of heifer calves
 Replacements born earlier
 Increase rate of herd expansion
 Select best cows to breed replacements from
 Fewer low value dairy bull calves
 Reduce difficult calvings and associated problems
 Improve biosecurity
Embryo transfer (ET) technology

Reproductive Anatomy
 What is an Embryo?

 An embryo is an egg that has

already been fertilized by a
sperm cell

• It is an organism in the earliest

stage of development
 What is Embryo Transfer?

• ET involves the removal of an embryo from a female of superior

genetics and the placement of the embryo into the reproductive tract
of a female of average genetics

What is the Goal of Embryo Transfer?

 The goal of ET is to obtain the maximum number of

genetically superior embryos in a minimum amount of
time
 Benefits of Embryo Transfer
• Traditionally, cows produce only one calf per year

• ET allows the production of many offspring within a year

from a single cow

• ET can increase the genetic potential of a herd in a relatively

short period of time

• ET can increase milk production in dairy herds

• ET can increase weaning weights in beef and dairy herds

• ET allows other producers to take advantage of superior

genetics because frozen embryos can be shipped almost
anywhere.

• ET preserves superior genetics for future generations due to

embryo freezing
 Necessary Equipment for Embryo Transfer

• Plastic media bag

• Foley catheter

• Embryo filter
 Necessary Equipment for Embryo Transfer (continued)

• Microscope

• Straw

• Rod
 Necessary Equipment for Embryo Transfer (continued)

• FSH
 Prostaglandin
(Lutalyse)
 Necessary Equipment for Embryo Transfer (continued)

• Penicillin • Lidocaine
 Necessary Equipment for Embryo Transfer (continued)

• Plastic Sleeve

• Bull Semen
 The Process of Embryo Transfer

 ET begins with the selection of a donor cow

• The donor cows will contribute the embryos to be transferred

 Donor Cows Have Superior Characteristics

 High milking ability

 High growth rate

 Outstanding reproductive capacity

 Bull Selection

• Next, a bull with superior • Breeding can occur naturally

genetics should be selected or by artificial insemination
 Recipient Cows

• Finally, recipient cows must be selected

• Recipient cows serve as surrogate (foster) mothers to the calves, but

contribute no genetic information

• For this reason, the genetic makeup of the recipient cow is not as
important as the makeup of the donor cow

• However, the recipient cow must be able to maintain her pregnancy

to term and produce an adequate milk supply for her calf
 Synchronizing the Estrous Cycle

 Once the donor and recipient cows have been selected,

 They must be synchronized so they are on the same phase of their

estrous cycle

 It is important to synchronize estrous cycles because the

reproductive environments of the donor and recipients must be
identical in order for the embryo to survive the transfer

• The estrous cycle is controlled by the production and secretion of

hormones at the proper time during the cycle

• Prostaglandin (PGF2α) is the hormone used to synchronize the

estrous cycles of the donor and recipient cows
 Synchronizing the Estrus Cycle (continued)

• Prostaglandin is produced naturally by the

cow

• However, a synthetic version called Lutalyse

is given in one or two injections to
synchronize estrous cycles
 Preparing the Donor Cow to be Flushed
 Before the donor cow is flushed, she is super ovulated with a
series of injections of Follicle Stimulating Hormone (FSH)
• Ovulation is the process of releasing eggs
• Superovulation causes the ovary (the female reproductive
organ) to produce many follicles
• Follicles are small blister-like structures that develop on the
ovary containing one egg each
• When the follicles ovulate, the eggs are released
• Superovulation ensures that many eggs will be released
because there are many follicles present
 Breeding the Donor Cow
• When the donor shows signs of estrus (the time period during the
estrous cycle when she will allow breeding), she is ready to be bred

• Some signs of estrus are riding other cows, clear vaginal mucus,
and pacing the fence

• If using artificial insemination, the donor cow should be bred at

least twice to ensure that all eggs are fertilized
 The Flush
 Once the donor cow has been bred, the embryos are allowed to
grow for six days
 During this time the embryos also travels down the reproductive
tract from the oviduct (the site of fertilization) to the uterus where
they can be flushed out
 On the seventh day, the embryos are ready to be removed. This
process is called flushing
 Embryo professionals use a non-surgical method to remove the
embryos. The process requires experience and a patient, steady
hand
 The Flush (continued)

• An injection of lidocaine is given prior to the

flush to reduce pressure and stress on the
donor cow and to make the flush easier for the
ET professional

• To begin the flush, a catheter is passed through

the cervix into one uterine horn
 The Flush (continued)

• The catheter contains a balloon that is

inflated with a saline solution in order to
seal the entrance to the uterus so fluid and
embryos are not lost
 Removing the Embryos

• The uterine horn is filled with

flush media and massaged to
allow the embryos to flow out of
the tract

• This process is repeated several

times in each uterine horn
 Collecting the Embryos

• Embryos are carried out of the

reproductive tract through plastic
tubes and collected in a filter with
the flush media

• The pores in the filter are smaller

than the embryos so excess fluid
drains out of the filter without
losing the embryos
 Injecting Penicillin

 After the embryos have been flushed out,

uterus injected with penicillin to kill any
missed embryos or infections
 Embryo Statistics

• An average of 7-10 embryos is collected from each flush

• However, the number of embryos obtained from a single flush

may range anywhere from 0-60
 Separating the Embryos

• In the lab, embryos are separated

from the flush media and
examined under a microscope to
determine their quality and stage
of development
 Embryo Size and Quality

• Embryos are microscopic in size (about 0.2 mm)

• Only undamaged embryos at proper maturity

should be transferred
 Embryo Quality

The embryo on the

The embryos on the left right is of proper
are damaged and should maturity and quality
not be transferred and should be
transferred
 Transferring the Embryos

• The embryo to be transferred is put into a small, plastic straw and

then loaded into an embryo transfer gun
 Transferring the Embryos (continued)

• The embryo is then inserted into

either the left or right uterine horn
depending on which ovary has a
corpus lutuem (CL)
• The CL is a structure on the ovary that
secretes the hormone progesterone
which is needed to maintain the
pregnancy
 Transfer Immediately or Freeze

• Embryos should be transferred as soon as

possible after the flush (within 8 hours at least)

• Embryos can also be frozen for later implantation

and stored in liquid nitrogen tanks
INVITRO EMBRYO PRODUCTION (IVEP)
 In vitro embryo production (IVEP)

Involves:

Collection of oocytes

In vitro maturation (IVM)

In vitro fertilization (IVF)

In vitro culture (IVC)

 Collection of oocytes

Live animal Slaughtered animal

Ovum pick-up Aspiration

 Collection of oocytes

ovaries collected from

slaughterhouse

Transport to lab in saline

containing antibiotics at 32-
37°C

Aspiration of follicles (2-8 mm) in

aspiration medium (TCM-199 + 10% FBS)
 Grading of oocytes

Grade A:Compact cumulus-oocyte-complexes (COCs) with an

unexpanded cumulus mass having ≥4 layers of cumulus cells, and
with homogenous evenly granular ooplasm

Grade B:COCs with 2-3 layers of cumulus cells and a homogenous

evenly granular ooplasm

Grade C: Oocytes partially or wholly denuded or with expanded or

scattered cumulus cells or with an irregular and dark ooplasm
 In vitro fertilization

In vitro matured oocytes Frozen semen

Thaw

In vitro sperm treatment and Capacitation

Incubation with 1-2 million/ml

spermatozoa for 18 h

Presumptive zygotes washed with IVC media

 Sperm treatment and Capacitation

Through washing and exposure in media

Enhance motility of spermatozoa

Express the acrosome reaction

Enhance successful fertilization of the oocytes

 Components of Capacitation media

Bovine serum albumin

Heparin - glucoseaminoglycans

Caffeine–cyclic nucleotide phosphodiesterase inhibitor

 In vitro culture of embryos

Presumptive zygotes washed with IVC media

Placed in 50-100 μl droplets of IVC media in 35 mm

Petri dish in groups of 10-15

Cultured for 9 days in IVC medium in a CO2 incubator

(5% CO2 in air,90-95% humidity) at 38.5°C
 Important consideration for IVMFC

Water quality

Buffering system and Osmolarity

Flux culture

Effect of maturation time

 Other consideration..

Antibiotic cover & mineral oil covering

Light environment

Temperature and gas phase

Storage and maintenance of media

 Culture supplements

Protein

Hormones and growth factors

Culture vessel
 In vitro culture system

Complex medium
Simple medium
with somatic cells
 Criteria to evaluate the quality of embryo

Excellent : Spherical, Symmetrical with cells of uniform size

Good : Few extruded blastomeres, irregular shape

Fair : Extruded blastomeres, few degenerated cells

Poor : Numerous extruded blastomeres, degenerated cells

 Evaluation of embryo development

2-16: cell stages

Morula : Embryo with >32 cell stage

Compact morula : Compaction of blastomeres

Early blastocyst: Formation of blastocoel had just started

Blastocyst : Well defined blastocoel & start differentiation

Expanded blastocyst : Expanded with zona thinning

Hatched blastocyst : partly or completely come out from ruptured

zona
 Chronology of embryo development

Cell stage Estimated age in days

Cattle buffalo
Morula 5 4
Compact morula 6 5
Early blastocyst 7 6
Blastocyst 7 6
Expanded blastocyst 8 7
Hatched blastocyst 9 7.5 to 8
 Stem cell Transgenic animal

Genome Cloning
reprogramming
Application of
IVEP
Early Cryopresevation
developmental
gene

Drug testing Embryo sexing

Cryopreservation Gametes (Haploid genome)
 Animal Genetics & Genomics

▪ Genetics: A branch of biology which studies heredity and variation in

organisms
▪ Genetics studies the transmission of genes from one generation to
another
▪ A blueprint of traits and characteristics is established for the new
offspring from the genes transferred from both parents
▪ The genotype is the genetic makeup, while the phenotype is the
physical makeup
 The relationship between phenotype and genotype is
expressed as the following equation:

P=G+E

P = Phenotype

G = Genotype, and

E = Environment
 Genes and Chromosomes
▪ Chromosomes (found in pairs) are contained in the nucleus of
every cell
 Within the chromosomes are smaller units called genes
▪ Genes contain the information that control all of the biochemical

processes (life processes) of the cell

 Chromosomes
▪ Organized structures of DNA
▪ Contained in the nucleus of the cell
▪ Found in pairs
▪ Numbers of pairs are species specific
▪ Humans – 46, Cattle – 60, Swine – 38,
Sheep – 54, Goats – 60, Horses – 78, Dog – 78,
Cats – 38
 Genes
▪ Genes contain the information that controls all of the biochemical
processes of the cell

▪ The gene codes are for the synthesis of specific proteins of the cell
 Genome
 The complete genetic material of an organism
▪ Genome bases are build to preserve species in as pure a state as
possible
 Gametes
▪ Male gametes are the sperm cells
▪ Female gametes are the egg cells
▪ They are the germ cells of reproduction
▪ Each normal body tissue cell (somatic cell) has 1 pair of
sex chromosomes
The other chromosomes within the somatic cell are called autosomes
▪ Somatic cell = Sex chromosome + autosome
 Haploid and Diploid Numbers

▪ All somatic cells contain a diploid (2n) number of chromosomes

▪ The germ cells, sperm and egg, contain a haploid (1n) number
 Inheritance
▪ The method by which alleles are passed from one generation to
another is called inheritance
▪ Gametes are produced from reproductive cells by the parent
▪ Each gamete contains a single allele for each gene or ½ of the genetic
code of the parent
 Genotypes and Phenotypes
▪ When gametes are combined during fertilization, a complete set of
genetic code is present

 This complete set contains the genetic traits of the new individual
is called the genotype of the animal

▪ The physical traits which are expressed from the genetic code
present or the physical appearance of the animal is called its
phenotype
What is Genomics?

▪ Study of how the genome (DNA) of any species is

organized and expressed as traits

▪ New technologies allow examination of an organism‟s

genome as a whole rather than 1 gene at a time

▪ Livestock and poultry genomes sequenced to

understand how various genes function (functional
genomics)
 How do we use genomics ?

▪ Identify DNA sequences associated with disease resistance and

production traits
▪ Animals can be evaluated as soon as DNA can be obtained (even
before birth)

▪ Best animals to be parents can be determined earlier and more

accurately
Embryogenesis:
 Formation of body structures & organs (organogenesis)
 Requires cell division (proliferation) and cell differentiation
(specialization)

Steps of Embryogenesis
1.Gametogenesis
2. Fertilization
3. Cleavage
4. Blastulation
5. Gastrulation Embryonic Development
6. Differentiation
7. Growth
Gametogenesis
 Is a process by which diploid or haploid cells undergo cell
division and differentiation to form mature haploid gametes (eggs
and sperm)
 Gamete formation where daughter cells, or gametes, are produced
at the end of meiosis II resulting in the production of sperm and egg
 Occurs by meiotic division of diploid gametocytes into various
gametes, or by mitotic division of haploid gametogenous cells

 Animals produce gametes directly through meiosis in organs

called gonads
 Males and females of a species that reproduces sexually have
different forms of gametogenesis:
▪ Spermatogenesis (male) in testes produce sperms
▪ Oogenesis (female) in ovary produce ova
 Types of Gamete Formation
Oogenesis: the process of
Spermatogenesis: the
process of male gamete female gamete production in

production in animals animals

Spermatogenesis process

 Spermatogenesis begins with diploid

germ cells called spermatogonia that
grow and develop into a primary
spermatocyte.

 During meiosis I, one spermatigonium

divides into two cells called secondary
spermatocytes.

 Secondary spermatocytes undergo

meiosis II to form spermatids, which
develop into sperm.

 In the end, from 1 parent spermatigonium

4 sperm cells are produced.
 Spermatogenesis occurs
inside testis

 Within testis spermatogenesis

occurs in walls of
seminiferous tubules

• Mature sperm are released

into lumen of seminiferous
tubules

• Spermatigonia & Primary

spermatocytes are 2n

• Secondary spermatocytes are

1n
• Spermatids and sperm are 1n
 Oogenesis process
• Oogenesis begins with a diploid cell called an
oogonium

• After growth and development, one oogonium forms

one primary oocyte

• Meiosis I produces a secondary oocyte and one polar

body (due to unequal division of the cytoplasm)

• Meiosis II in the secondary oocyte produces an egg

and polar body

Meiosis II in first polar body produces 2 polar bodies

• The egg survives, while all the polar bodies die
• Only one functional egg cell comes from this
process, as the unequal division of the cytoplasm
makes the egg cell big (needs extra nutrients)
Oogenesis occurs inside the ovary
• Oogenesis occurs in a follicle
• Eggs are released into oviduct
• Oogonium & Primary oocytes are 2n
• Secondary oocytes are 1n
• Polar bodies and eggs are 1n
THANK YOU!!!