TRANSGENIC ANIMALS

TRANSGENIC ANIMALS

24.      TRANSGENIC ANIMALS

A transgenic animal is one that carries a foreign gene (usually belonging to a different species) that has been deliberately inserted into its genome. The foreign gene called transgene is constructed using Recombinant DNA technology. The first transgenic animal was a "Supermouse" created by Ralph Brinster and Richard Palmiter in 1982. It was created by inserting a human growth Hormone gene in the mouse genome. The offspring was much larger than the parents. The mouse is common transgenic experiment other animals include pig, cow, sheep etc.
i.    It's a powerful tool for studying gene expression and developmental process in the higher organism.
ii.    A good model for the understanding of the disease.
iii.    Transgenesis is important for improving the quality or quantity of milk, eggs, meat and wool production beside coacting drug resistant animal.
Limitation :
i.    Transgene may not be expressed or sometime over expressed.
ii.    It's very time consuming, costly and labour expensive.
24.1.    Production of Transgenic Animals – The methodology
Step 1    Construction of a transgene. Transgene made of 3 parts.
i.    Promoter
ii.    Gene to be expressed        
iii.    Terminator sequence
Step 2    Introduction of the foreign gene into the animal. Three 3 methods are usually follows-
i.    Pro nuclear micro injection method.
ii.    Embryonic stem cell method.
iii.    Retro viral vector method.
24.1.1.    Pronuclear Microinjection Method

24.1.2.    Embryonic stem cell method 
Transgenic animals can be created by manipulating embryonic stem cells.

The developing embryo is a Chimera, that is consists of two populations of cells derived from different zygotes, those of the blastocyst and the implanted ES cells. This differs from a mosaic in which the cells may be genetically different but are derived from the same zygote. The desired genetically engineered cell with transgene can be identified by using a marker gene or PCR analysis.
24.1.3.    Retroviral Vector Method
Retroviral vectors are used as an effective means of integrating the transgene into the genome of a recipient cell. However, these vectors can transfer only small pieces (~8kb) of DNA.
Step 3    Screening for the transgenic positive organism.
Transgenic progenies are screened by polymerase chain reaction (PCR) to examine the site of incorporation of the gene.
Some transgene may not be expressed if integrated into a transcriptionally inactive site of the genome.
Step 4    Further animal breeding is done to obtain maximal expression.
Heterogenous offsprings are mated to form homozygous strains.
24.1.4.    Importance of Transgenic Animals
1.    Medical Importance
        –    Disease model
        –    Bioreactors for pharmaceuticals
        –    Xenotransplantation.
2.    Agricultural Importance.
        –    Disease resistant animals
        –    For improving quality and quantity of milk, meat, eggs and wool production.
    3.    Industrial Importance.
        –    Toxicity sensitive transgenic animals to test chemicals.
        –    Spider silk, milk of the goat.
24.2.    Gene Knockout
A gene knockout is a genetically engineered organism that carries one or more genes in its chromosomes that has been made inoperative. The primary use of this technique has been to replace a normal gene with a mutant allele or a disrupted form of the gene, thus knocking out the gene's function.
Other forms of gene disruption:-
Gene knockdown – reduces the expression of the gene.
Knockin – replace one allele (eg. wild type) with a functional another allele (eg. a specific mutation)
There may also be simultaneously knocking out Two genes knock out is known as a double knock out and three genes knock out is the triple knock out.
24.2.1.    The process of developing knockout mouse:-

24.2.2.    Applications of gene knockout:-
To determine the function of gene products.
To create mouse modal of human genetic diseases.
To characterize genetic regulatory regions.
24.2.3.    Limitations:-
Genetically altered embryos cannot grow into adult mice.
The lack of adult mice limits studies to embryonic development.
The gene may serve a different function in adults in developing embryos.
24.2.4.    Formation & selection of recombination ES Cells:-
Antibiotic resistance genes are inserted along with desired genes into the ES cell for the identification of ES cells that have been transfected. The desired gene near, a gene that encodes an enzyme that inactivates the antibiotic neomycin and its relatives, like the drug G418, which is lethal to mammalian cells; tk, a gene that encodes thymidine kinase, an enzyme that phosphorylates the nucleoside analogue gancyclovir.
DNA polymerase fails to discriminate against the resulting nucleotide and inserts this non-functional nucleotide into freshly replicating DNA. So gancyclovir kills cells that contain the tk gene.


Results:-
The cells (the majority) that failed to take up, the vector are killed by G418.
The cells in which the vector was inserted randomly are killed by gancyclovir (because they contain the tK gene). This leaves a population of cells transformed by homologous recombination (enriched several thousand fold).


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