HISTORY AND TOOLS IN GENETIC ENGINEERING

History
 Father of Genetic Engineering is Paul Berg. He was the first who developed recombinant DNA
technology

The Beginning
 The first genetically modified animals was mouse created in 1973 by Rudolf Jaenisch
 In 1993, an antibiotic resistant gene was inserted in tabacco plant, leading ot first genetically modified
plant
 In 1978, the technology was commercialized with the production of insulin
 In 1994, first genetically modified food, tomato, was made

Definition
 Change in genetic make up of living cells by inserting desired gene through a vector–Genetic
Engineering.
Terms
Gene
 small piece of DNA that encodes for a specific protein
Recombinant DNA
 DNA formed by joining DNA segment of two different organism
Recombinant DNA technology
 technique by which gene of interest is transferred to the host
Genetically Modified Organism
 organism whose genetic makeup is altered/changed using rDNA technology

Procedure
1. Isolation of desired DNA fragment (gene of interest) with the help of restriction enzymes
2. Isolation of DNA vector
3. Construction of rDNA. In this, gene of interest is inserted into the vector
4. Introduction of vector containing recombinant into the host cell
5. Multiplication of Host cells containing recombinant DNA
6. Expression of cloned gene
7. Selection of Recombinant cells

TOOLS USED IN GENETIC ENGINEERING

1. Restriction Endonuclease (RE)
 Enzyme which cleaves the DNA from particular sequence
 Sequence from where it cleaves the DNA is called as Recognition Sequence
- Recognition site can be 4-8 bp long
 Breaks the nucleotide bond of base pair
Types of Restriction Endonuclease
Type I
 Made up of three non-identical subunits
 Require ATP, mg2+ for activation
 Cleave the DNA 1000 bp away from the recognition
Type II
 Requires only mg2+
 Made of two identical subunits
 Cleaves DNA from recognition site
 Widely used enzyme
 More than 300 enzymes are discovered
Type III
 Cleave 26 bp away from recognition site
 2. Gene Library
 The gene of interest is stored in gene library
 There are two gene libraries available
Genomic library
 Collection of clones containing all DNA segments of the genome of an organism is called Genomic
library
cDNA
 Collection of clones each of which carries a cDNA of an organism

3. Vector
  DNA molecule that has the ability to replicate autonomously in a host cell into which the DNA fragment
to be cloned
 Any extra chromosomal small genome/DNA, self-replicating e.g. Plasmid (pBR322, pUC18/19), Phage
( phage, phage M13), Cosmid, Phasmid, BAC, YAC

Properties of Good Vector

 Should have origin of replication
 Should be less than 10 kb in size
 Easy to isolate and purify
 Easily introduced to host cell
 Should contain unique target site for many RE

4. Host Cell
 Organism in which rDNA are to be transformed
 Best example for host cell is E. coli. (Escherichia coli)

Properties of Good Vector

1. Easy to transform
2. Support the replication of rDNA
3. Lack active restriction enzyme

Gene Transfer Method

 The transfer of rDNA into a bacterial cell or plant cell or animal cell
 The host cell contains an rDNA is known as transformed cell or recombinant
 The rDNA in cell replicates independently of the chromosomal DNA of cell
 The desired foreign gene present in the rDNA express its characters in host cell
Method
In Prokaryotes:
 Transformation
 Transduction
 Electroporation
In Eukaryotes:
 Transfection
 Electroporation
 Ultrasonication
 Bombardment
 Microinjection
In Vitro Production of Insulin
 Isolation of gene responsible for insulin production
 Isolation of plasmid
 Gene insertion in plasmid
 Introduction of rDNA in host
 Multiplication of recombinant host cell
Transgenic Plant
The plants that have been genetically engineered
 Herbicide resistance
 Insect resistance
 Virus resistance
 Improved storage
 Environmental stress resistance
 Improved nutritional quality
Example: BT Cotton
 Genetically Modified Organism cotton variety, which produce insecticide to bollworm

Transgenic Animal
 Genetically manipulated animal having an introduced gene
 Transgenic mice, sheep, cattle, goats, pigs, poultry, and fishes have been developed using GE
Example: Glofish
 Genetical modified fluorescent fish
 Done by bioluminescence, found in fireflies or lightning bugs
 An enzymes called as luciferase is taken from firefly and is inserted fishes to make them glow
Application
Agriculture
  Improved crops
 High yield
 Resistant
 High nutritional value
 Long storage
Medicine
 Production of insulin and human growth hormone
Animal husbandry
 High milk production
 High yield of wool