Professional Documents
Culture Documents
Bikash Karki
Shrawan Shah
Submitted TO Dines S. Pujara
Anup Sharma
IVth Semester
Bsc.
Biotechnology
SANN college
Presentation overview
# Introduction To GMO
# Transgenic Animal
# History
# Applications Of Transgenic Animals
#Methods To Produce GMO
# Knockout Mouse Project
# Application of KOMP
# Myths And Ethics
# Conclusion
# References
WHAT ARE GMO or TRANSGENIC ANIMALS ?
Transgenic Animals
A transgenic animal is one that carries a foreign gene that has been
deliberately inserted into its genome. The foreign gene is constructed using
recombinant DNA methodology. In addition to a structural gene, the DNA
usually includes other sequences to enable it
► to be incorporated into the DNA of the host and
► to be expressed correctly by the cells of the host.
History of transgenic animal production:
The term “GMO" does not always imply targeted insertions of genes
from one into another species For example, a gene from a jellyfish,
encoding a fluorescent protein GFP, can be physically linked and co-
expressed with mammalian genes to identify the location of the protein.
Transgenic microbes
Medical
to produce the protein insulin, in treatment of treat diabetes
to produce clotting factors to treat haemophilia
human growth hormone to treat various forms of dwarfism
genetically modified viruses allow gene therapy , is being developed for
a treatment of incurable diseases, such as cystic fibrosis,
sickle cell anemia and muscular dystrophy
is also used in some soils to facilitate crop growth, and can also produce
chemicals which are toxic to crop pests
During the 1970s, the first chimeric mice were produced (Brinster, 1974)
Cells of two different embryos of different strains were combined together at an
early stage of development (eight cells) to form a single embryo that subsequently
developed into a chimeric adult, exhibiting characteristics of each strain.
DNA microinjection, (Gordon and Ruddle ),1981
the first technique being proved successful in mammals, was first applied to mice
and then to various other species such as rats, rabbits, sheep, pigs, birds, and fish
the term transgenic was first used by J.W. Gordon and F.H. Ruddle (1981)
Two other main techniques were then developed
those of retrovirus-mediated transgenesis (Jaenisch, 1976) and embryonic stem
(ES) cell-mediated gene transfer (Gossler et al., 1986).
Methods of creation of transgenic animals
Direct microinjection
Virus mediated gene transfer
Nuclear transfers
Sperm-mediated gene transfer
Artificial chromosomes for gene transfer
Embryonic stem cells
(KNOCKOUT MOUSE)
Direct microinjection
• inject DNA molecules (transgenes) directly into male pronucleus
• manipulated fertilized ovum is transferred into the oviduct of a recipient female, or
foster mother
• most popular technology, commercial available
• the success rates range from 10-30% depending on skills and constructs
• the insertion of DNA is a random process, and there is a high probability that the
introduced gene will not insert itself into a site on the host DNA that will permit its
expression
• the efficiency is not related to the copies of transgenes injected
• initial investment is high, a minimum of $100K to start
Virus Mediated Gene Transfer
• creation of Dolly
• somatic cells be transfected, or genetically altered prior to NT
• 100% efficiency of any progeny
• abnormal development
WHAT IS DOLLY????
The Embryonic Stem Cell Method
Stem cells are undifferentiated cells that have the potential to differentiate into any
type of cell.
These cells are incorporated into an embryo at the blastocyst stage of
development.
Embryonic stem cells (ES cells) are harvested from the inner cell mass (ICM) of
mouse blastocysts.
Structural gene of desire is inserted OR knocked out via DNA-Recombination.
Successfully transformed cells are selected and injected into inner cell mass of
mouse blastocyst.
Embryo transfer
Prepare a pseudopregnant mouse (by mating a female mouse with a vasectomized
male). The stimulus of mating elicits the hormonal changes needed to make her
uterus receptive.
Transfer the embryos into her uterus.
What is a knockout mouse?
Knockout mice are important animal models for studying the role of genes which have
been sequenced, but have unknown functions. By causing a specific gene to be
inactive in the mouse, and observing any differences from normal behaviour or
condition, researchers can infer its probable function.
Mice are currently the most closely related laboratory animal species to humans, for
which the knockout technique can easily be applied.
The Nobel Prize in Physiology or Medicine 2007
"for their discoveries of principles for introducing specific gene
modifications in mice by the use of embryonic stem cells“
is awarded to..
The first knockout mouse was created by Mario R. Capecchi, Martin Evans and Oliver
Smithies in 1989, for which they were awarded the Nobel Prize for Medicine in 2007.
PRODUCTION OF KNOCKOUT MICE.
Harvesting of ES cells
Depending upon methods of insertion of artificial DNA into the chromosome of ES
cells nuclei there are TWO METHODS TO PRODUCE KNOCKOUT MOUSE. Both
methods are carried out in vitro.
Chimeric F1 mice
For both gene targeting and gene trapping, a modified viral vector or a linear
fragment of bacterial DNA is used to insert the artificial DNA into ES cell.
After insertion, the genetically altered ES cells are grown in a lab dish for several
days and injected into early-stage mouse embryos.
The embryos are implanted into the uterus of a female mouse and allowed to
develop into mouse pups.
The resulting mouse pups are not complete knocked out, that is with both normal
& altered ES cells. These are crossbreed to produce lines of mice in which both
copies of the gene are knocked out in all tissues, called homozygous knockouts.
Knockout mice gives information that can be used to better understand how a similar
gene may cause or contribute to disease in humans.
Atherosclerosis
Inherited Heart Disease
Monogenic Disease Complex Disease
Lesch-Nyhan syndrome
Essential hypertension
Cystic fibrosis Atherosclerosis
inherited heart diseases
Cancer
Other diseases
Obesity, Diabetes, Arthritis, Substance abuse, Anxiety, Aging and Parkinson's disease.
Gene Targeting In Disease Diagnosis And Study
Cystic fibrosis, Defective chloride transport through cAMP-activated chloride channel in mouse
due to knocking out of cystic fibrosis transmembrane conductance regulator (CFTR) gene.
The similar phenotypes is observed in human as in mice.
Agricultural Applications
Breeding;
Quality
Disease Resistance
Medical Application
Xenotransplantation
Nutritional Supplements and Pharmaceuticals
Human Gene Therapy
Industrial Application
Nexia Biotechnologies in Canada transformed spider gene in goat & it began to secrete tiny
silk strands from their body. By extracting polymer strands from the milk and weaving them
into thread, the scientists can create a light, tough, flexible material that could be used in such
applications as military uniforms, medical microsutures, and tennis racket strings.
Ethical concerns surrounding transgenesis
THANK