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Animal Biotechnology. Animal Biotech. Animals provide a number of products we use in every day life: Milk Leather Wool Eggs Meat. Animal Biotechnology. Transgenics Characterizing genetic variability Reproductive technologies Artificial insemination (AI) and preservation of semen
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Animal Biotech • Animals provide a number of products we use in every day life: • Milk • Leather • Wool • Eggs • Meat
Animal Biotechnology • Transgenics • Characterizing genetic variability • Reproductive technologies • Artificial insemination (AI) and preservation of semen • Improving health through developing vaccines • Diagnostics and epidemiology • Nutrition and feed utilization
Transgenics • Transgenics are genetically modified organisms with DNA from another source inserted into their genome • A large number of transgenic animals have been created Mice Cows Pigs Sheep Goats Fish Frogs Insects • Currently, no transgenic animal or animal product is approved by the FDA or USDA for human consumption
Goals of transgenic animal creation • Improve livestock animals • Genetic modification of animals may be aimed at improving their milk, meet, wool etc production. • Use of animals as bioreactors • Genes are transferred into the animals to obtain a large scale production of proteins encoded by these genes in milk, urine or blood of such animals; such animals are called bioreactors and this method is called molecular farming or gene farming.
Research into animal and human disease • A special case of gene transfer is alleviating or eliminating the symptoms and consequent miseries of genetic diseases. • Specific transgenic animals strains or lines are created to fulfill specialized experimental and/or biomedical needs. Eg. Konck out mice strains in which specific genes have been replaced or knocked out by their disrupted counterparts through a process of homologous recombination.
Making a transgene • Structural gene which selected as per requirement • Vectors;are carriers used to transfer the gene of interest from one organism to another. Eg. SV40 vectors, bovine papilloma vectors, retrovirus vectors, bacculovirusvectors • Requirements of promoter/ enhancerseq in the transgene so that it can be expressed in the host cells. • A transgene must be integrated into the host genome for obtaining transgenic cells/animals. • Transgene must be present in proper orientation in relation to and in association with various sequences required for its efficient transcription and translation in host cell.
Gene Constructs • A transgene must be integrated into the host genome for obtaining transgenic cells/animals. • Transgene must be present in proper orientation in relation to and in association with various sequences required for its efficient transcription ans translation in host cell. The sequences may be listed as follow; • An efficient promotors/ enhancer • The translation initiation codon (AUG in mRNA, and ATG in DNA) • The chain termination codon • Transcription termination sequence • Polyadenylation cleavage/ addition site
Gene Construct Schematic representation of a gene construct for expression in animal/ plant cell. P/E ATG Gene TAG Poly- (A)
Fully transgenic organisms Non transgenic organisms Mosaic organisms Construction of Transgenic Animal Isolation of transgene Introduction into cell Integration of gene into the genome Development of organism Insertion into vector
Creation of Transgenic Animals by Nuclear Injection • In vitro fertilization is used to start a transgenic animal. • Harvested eggs and sperm are fertilized, and before the pronuclei fuse, the transgene is injected into the male pronucleus. • The embryo continues to divide in culture and is then implanted into a mouse. • The “foster mother” mouse has been treated with hormones so that she accepts the embryo and carries on with the pregnancy. • The offspring are screened for stable integration of the transgene. • Founder mice have one copy of the transgene. Gene injected into the male pronuclei
Detection of Transgenics and Transgene Function • Dot Blot technique • PCR amplification using genomic DNAs extracted from mice • Detection of mRNA Expression • Assays for protein Expression
Large Transgenic Mice • Express the rat somatotropin gene • Under the control of the promoter from an unrelated mouse gene, metallothionein , which is normally expressed in the liver • Instead of being made in the pituitary gland, the normal site for growth hormone, the rat somatotropin is manufactured in liver • Human somatotropin has also been expressed in mice and also gives bigger mice. • Dwarfism • Recombinant human somatotropin (rHST) is now used to treat the hormone-deficient type of dwarf.
Large Transgenic Mice • Express the rat somatotropin gene • Under the control of the promoter from an unrelated mouse gene, metallothionein , which is normally expressed in the liver • Instead of being made in the pituitary gland, the normal site for growth hormone, the rat somatotropin is manufactured in liver • Human somatotropin has also been expressed in mice and also gives bigger mice.
Trendy Transgenic Mice • Marathon Mouse • Can run about 1800 meters—more than a mile—before exhaustion. This is twice as far as a normal mouse can last. • Marathon mouse has enhanced PPAR-delta—a regulator of several genes involved in burning fat and in muscle development. • Mighty Mouse • Lack myostatin, a protein that slows muscle growth. The result is colossal muscle development. • There are one-two known cases of a human with a genetic defect leading to lack of myostatin. A German boy, born in Berlin in 2000, has muscles twice the size of other children his age.
Recombinant protein production • The somatotropin gene from Cow • Can be expressed in bacteria - the production of large amounts of the hormone, rBST. • Used in the dairy industry to increase milk production. • Used to boost adult cow’s somatotropin, resulting in increased milk production. • If cloned in Cow? • Production of heterologous proteins • Recombinant proteins can be produced in the milk from transgenic cows or other farm animals. • The cloned genes are placed under the control of a regulatory region that will allow gene expression only in the mammary gland. • For small-scale production, transgenic goats are often used. • E.g. transgenic goats producing rTPA
Knockout Mice • The target gene is cloned and disrupted by inserting a DNA cassette. • This work is usually done in bacteria. • Once the construct is made, it is put back into a mouse by injection into the male pronucleus during fertilization. • After the transgenic offspring are born, two heterozygotes are crossed to create a homozygous knockout mouse. • These are then screened for defects due to inactivation of the target gene.
