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TRANSGENIC ANIMALS Presented by S haranabasappa I st M.Pharm. Dept. of Pharmacology Subject In Charge SYED MANSOOR AHMED M.Pharma.(Ph.D) Dept. of Pharmacology Sree Siddagagnga College of Pharmacy , Tumkur.


CONTENTS Introduction Commercial interest in TAs Purpose Methods of production Applications of transgenesis Nude mouse References


INTRODUCTION A transgenic animal is one which carries a foreign gene that has been deliberately inserted into its genome. The foreign gene is constructed using rDNA technology. The animals whose genetic composition has been altered by the addition of foreign DNA is said to be transgenic. The DNA that is introduced is called as a transgene & the overall process is called transgenic technology or transgenesis.

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Continued …… Another method is to knock out or disrupt one of the animals’ own genes; scientists call these animals “Knockouts”. These are usually used in biomedical research where they mimic a human disease. Transgenic animals are able to pass on their new genes to their offsprings thereby producing future generations of modified animals.

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Continued …… The first transgenic animal was created nearly 20 years ago and it was a giant mouse which had the gene for human GH incorporated into its genome. Since then transgenic rats, rabbits, sheep, cows, pigs, goats, chickens & fish have been designed.

Commercial interest in transgenic animals:

Commercial interest in transgenic animals Broadly for two reasons For conventional products in more quantity. Ex:- meat, milk, leather, wool etc. For synthesis of non-conventional products i.e. animals as bioreactors to produce pharmacologically valuable proteins. Ex:- Interferon, α -antitrypsin etc



Methods of producing transgenic animals:

Methods of producing transgenic animals For gene transfer in animals, 3 different techniques are possible: microinjection of DNA into the pronuclei of zygotes; DNA transfer using retroviral vectors; Embryonic stem cell-mediated gene transfer.

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Microinjection method Pseudopregnant recipient Offspring Transgenic founder Pronuclear microinjection Embryo transfer Natural Birth Genotyping in vitro culture

Microinjection method:

Microinjection method Microinjection of DNA into pronucleus of fertilised egg. Procedure:- Young virgin female mice (4-5 weeks age) are hormonally induced to superovulate & then mated. 22 hours later they are sacrificed & oviducts are removed into buffered salt solution. Oviducts are dissected to release fertilised eggs. Eggs are washed & kept at 37 o C in culture medium. Eggs are observed under microscope to distinguish two pronuclei. Male pronucleus is larger than female pronucleus so it is chosen for injection. 40-60 eggs are injected in one hour, which are stored in culture medium. Many eggs may lyse.

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Continued …… 10-20 fertilised eggs (embryos) which have been gene manipulated (microinjection) are reintroduced into pseudopregnant female reproductive tract. “Tail-blot” is carried out to detect the presence of foreign DNA in offspring. (Blood is collected from the tail, DNA is prepared & probed to check the presence of specific introduced sequences) 25% of the surviving injected embryos are transgenic. These are founder animals.

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Continued …… Advantages Stable integration Rapid Method is relatively straightforward Disadvantages Efficiency is low (~10%) Site of integration is random & is variable in individuals Number of copies which integrate is highly variable Exact replication is difficult

Retroviral vector method:

Retroviral vector method Retroviral infection of fertilised egg at 4-8 cell stage Transformation of mouse embryos (4-8 cell stage) is done by infecting the cells of preimplantation mouse embryo with SV 40 or retrovirus ( such as MoMuLv). This infection can be done by Co-cultivating 4-8 cell pre-embryos with virus producer cell, or By injecting virus into blastocyst in vitro Infected pre-embryos are returned to pseudopregnant recipients & allowed to develop. Protective proteoglycan coat called Zona Pellicula which surrounds the blastocyst is first removed. The retrovirus integrates as a single copy & if germline cells are infected then the infected provirus is transmitted as Mendelian trait.

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Continued …… Advantages Effective & efficient expression Disadvantages Maximum only 8 kb of exogenous DNA can be inserted in retrovirus Retrovirus sequences may affect transgene expression Out breeding of founder animal is necessary to establish pure lines MoMuLv becomes methylated & expression blocked Integrated genome is expressed but in tissue specific manner i.e. different for different integrates

Embryonic stem cell method:

Embryonic stem cell method Transforming ES cells growing in tissue culture with the desired DNA. ES cells are harvested from the inner cell mass (ICM) of a mouse blastocysts. They can be grown in culture & retain their full potential to produce all the cells of the mature animals, including its gametes.

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Continued …… 1. Make your DNA Using recombinant DNA methods, build molecules of DNA containing the structural gene you desire (e.g., the insulin gene) vector DNA to enable the molecules to be inserted into host DNA molecules promoter and enhancer sequences to enable the gene to be expressed by host cells 2. Transform ES cells in culture Expose the cultured cells to the DNA so that some will incorporate it. 3. Select for successfully transformed cells.

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Continued …… 4 . Inject these cells into the ICM of mouse blastocysts. by Electroporation or Microinjection 5. 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. Hope that they implant successfully and develop into healthy pups (not more than one-third will). 6. Test her offspring Remove a small piece of tissue from the tail and examine its DNA for the desired gene. No more than 10–20% will have it, and they will be heterozygous for the gene.

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Continued …… 7. Establish a transgenic strain Mate two heterozygous mice and screen their offspring for the 1:4 that will be homozygous for the transgene. Mating these will found the transgenic strain.

Applications of transgenesis:

Applications of transgenesis Transgenic laboratory animals would be useful as animal model of human disease Ex:- * Animal model for cancer is Oncomouse. * Alzheimer's mouse is very useful in understanding the pathological basis of the disease. Transgenic animals would be useful for ph. Testing & development Useful for exploring techniques foe gene therapy – to cure genetic disorders Transgenic animals would have improved physiology so livestock production will improve Ex:- Transgenic sheep with increased wool production.

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Continued …… Useful as bioreactors to produce pharmacologically imp. Proteins. T. animal Protein product Biological Imp. Cow Lactoferrin In iron deficiency anaemia Cow Interferon In viral infections Sheep Alpha 1-antitrypsin In emphysema Goat Antithrombin III Regulates blood clotting Mouse Urokinase For dissolving blood clots Pig Haemoglobin Blood transfusion Rabbits Alpha- glucosidase Pompe’s disease

Nude Mouse :

Nude Mouse

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Continued ….. A mutant mouse is said to be nude because it is hairless due to the presence of two copies of gene “nu” (for nude). Discovered in 1962 by Dr. N.R.Grist at Ruchill Hospital’s Brownlee Virology laboratory in Glasgow. Life span:- normally 6 months to a year Mainly used as an animal model in immune deficiency forms, leukemia, solid tumours, AIDS, leprosy.


References Biotechnology by Dr. U. Satyanarayana Pg. no. 480-493 Gene biotechnology by S.N.Jogdana Pg. no. 250-259 Pharmaceutical biotechnology by J.A.Crommelin & Robert D Sindelar Pg. no. 140-145

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