Transgenesis in animals

Introduction
History
Landmarks Events in Transgenic Livestock Research
Techniques/ Method for Gene Transfer
Examples of transgenesis
Importance
Application
Limitation
Issue related to Transgenic Technology
Ethical concerns and how to Overcome

1. REPRODUCTIVE BIOTECHNOLOGY Dr. Dhaval Chaudhary M.V.Sc Scholar Dept. of Animal Genetics and Breeding College of Veterinary Science and Animal Husbandry, AAU, Anand Transgenesis

2. Content Introduction History Landmarks Events in Transgenic Livestock Research Techniques/ Method for Gene Transfer Examples of transgenesis Importance Application Limitation Issue related to Transgenic Technology Ethical concerns and how to Overcome

3. Introduction Process of introducing foreign or exogenous DNA into an animals genome is called transgenesis . Transgenesis is the process of introducing an exogenous gene called a transgene into a living organism so that the organism will exhibit a new property and transmit that property to its offspring . Transgenesis can be facilitated by liposomes by liposomes, enzymes, plasmid vectors, viral vectors, pronuclear injection, protoplast fusion, and ballistic DNA injection.

4. Definitions Transgenesis The stable, one or more integration of foreign genes /foreign DNA into a host’s chromosomes. OR Transgenesis either means transferring DNA into the animal or altering DNA of the animal. Transgenic animal A transgenic animal is one that carries a foreign gene that has been deliberately inserted into its genome. OR Transgenic animal are genetically modified to contain a gene from a different species following gene transplantation or resulting from the molecular manipulations of endogenous genomic DNA

5. History Prior to the development of molecular genetics, the only way of studying the regulation and function of mammalian genes was through the observation of inherited characteristics or spontaneous mutations. The discovery of DNA and genes opened wide avenues for research and biotechnological applications. The introduction of isolated genes into cells became a common practice in the 1970s, soon after the emergence of the genetic engineering techniques.

6. During the 1970s, the first chimeric mice were produced. It represented a great progress for the understanding of gene function and mechanisms of action. The first transgenic animal i.e. mice, were obtained by microinjecting the genes into one of the nuclei ( pronuclei ) of one day old embryos.

7. This construct was injected into fertilized mouse embryos and the resulting transgenic offspring, were fed with extra zinc, which turned on the metallothionin promoter. This resulted in the expression of growth hormone gene and the resulting high levels of circulating rat growth hormone dramatically changed the phenotype of the transgenic mice by stimulating them to grow twice as large as normal.

8. The giant mice instilled major excitement in the scientific and public communities, markedly enhancing attention on the transgenic mouse system. Ralph L. Brinster and Richard Palmiter thus were pioneered in the development of methods to transfer foreign genes into the germline of animals. PALMITER, R.D., BRINSTER, R.L., HAMMER, R.E., TRUMBAUER, M.E., ROSENFELD, M.G., BIRNBERG, N.C. and EVANS, R.M. “Dramatic growth of mice that develop from eggs microinjected with metallothionin -growth hormone fusion genes.” Nature (1982) 300: 611-615.

9. This technique is still widely used, gene transfer into animals and plants to generate lines of genetically modified organisms, known as transgenic animals and plants, respectively . This method could be extrapolated (extend the application of to an unknown situation by assuming that existing trends will continue successfully) to other mammals in 1985. Other transgenic animals include rats, pigs and sheep etc . Transgenic technology led to the development of fish that enabled to grow faster and livestock that enables to fight diseases (prion-free cows resistant to bovine spongiform encephalopathy, known as mad cow disease).

10. Two other main techniques were Subsiquently developed: those of retrovirus-mediated transgenesis ( Jaenisch , 1976) and embryonic stem (ES) cell-mediated gene transfer ( Gossler et al., 1986). The term transgenic was first used by J.W. Gordon and F.H. Ruddle (1981). The transgenic technology also became an excellent tool in basic research for understanding the functions and regulations of a number of mammalian genes.

11. Thanks to the transgenic technology, because today we have mouse models for several types of cancer and of human genetic disorders including chronic hepatitis, diabetes, Alzheimer's disease and many more.

12. Why Transgenic Animals Produced? To help scientists to identify, isolate and characterise genes in order to understand more about their function and regulation. To provide research models of human diseases, to help develop new drugs and new strategies for repairing defective genes (“gene therapy ”). To provide organs and tissues for use in human transplant surgery. To produce milk which contains therapeutic proteins; or to alter the composition of milk to improve its nutritional value for human infants. To enhance livestock improvement programmes.

13. Strategies for Producing Transgenic Animals There are two basic strategies for producing transgenic animals, which include “gain of function” or “loss of function” transgenics . The basic idea behind the gain of function strategy is that by adding a cloned fragment of DNA into an animal’s genome to a new gene product is produced that did not previously existed in that cell or tissue. E.g. expression of rat growth hormone in mouse and to get over expression of gene product in the proper tissue ( Palmiter et al., 1982). The silencing or loss of gene function is accomplished by the target gene disruption through the process of homologous recombination between host genome and exogenous DNA.

14. Techniques / Methods of Gene Transfer There are many techniques, all are listed below 1 ) DNA Microinjection 2 ) Retro virus- Mediated Gene Transfer 3 ) Embryonic stem cell / chimeras 4) Somatic Cell Nuclear Transfer / Cloning 5) Use of Transposons 6) Sperm Mediated Gene Transfer 7) Electroporation 8 ) Chemical technique 9) RNA Interference Some of important methods have been described in Houdebine 2003, 2005.

15. Houdebine 2003 , 2005 Different methods to generate transgenic animals: (1) DNA transfer via direct microinjection into pronucleus or cytoplasm of embryo ; ( 2) DNA transfer via a transposon: the foreign gene is introduced in the transposon which is injected into a pronucleus ; ( 3) DNA transfer via a lentiviral vector: the gene of interest in a lentiviral vector is injected between the zona pellucida and membrane of the oocyte or embryo; (4) DNA transfer via sperm: sperm is incubated with the foreign gene and injected into the oocyte cytoplasm for fertilization by ICSI (intracytoplasmic sperm injection); ( 5) DNA transfer via pluripotent cells: the foreign gene is introduced into pluripotent cell lines (ES: embryonic stem cells: lines established from early embryo, EG: embryonic gonad cells: lines established from primordial germ cells of foetal gonads); the pluripotent cells containing the foreign gene are injected into an early embryo to generate chimeric animals harbouring the foreign gene DNA ; ( 6) DNA transfer via cloning: the foreign gene is transferred into a somatic cell, the nucleus of which is introduced into the cytoplasm of an enucleated oocyte to generate a transgenic clone. Methods 1, 2 , 3 and 4 allow random gene addition whereas methods 5 and 6 allow random gene addition and targeted gene integration via homologous recombination for gene addition or gene replacement including gene knockout and knocking.

16. DNA Microinjection Gordon and Ruddle , 1981 The young virgin mice (4-5weeks age) are subjected to ovulation Administration of FSH (pregnant mare’s serum) After 2 days admistration of HCG Super ovulated mice produce 30-35 eggs Above female mice mated with males Fertilized eggs are removed from the fallopian tubes By micromanipulation using microinjection needle &holding pipette the DNA is injected into the male pronuclear of the fertilized egg Trans genes are kept overnight in an incubator The foster mother delivers pups after 3 weeks of implantation

17. Retro Virus- Mediated Gene Transfer Jaenisch , 1976 The transfer of small pieces (8kb) of DNA can be effectively carried out by retroviruses This method however is unsuitable for transfer of larger genes Further even of small genes there is a risk: losing some regulatory sequences . Drawback:-Risk of retroviral contamination in the products(in foods for human consumption). Not regularly use for transgenesis

18. Embryonic Stem Cell- Mediated Gene Transfer Gossler et al., 1986 This method involves prior insertion of the desired DNA sequence by homologous recombination into an in vitro culture of embryonic stem (ES) cells. These cells are then incorporated into an embryo at the blastocyst stage of development. ES cell-mediated gene transfer is the method of choice for gene inactivation method (knock-out method). This technique is of particular importance for the study of the genetic control of developmental processes. This technique works particularly well in mice. It has the advantage of allowing precise targeting of defined mutations in the gene via homologous combination

19. DNA transfer via C loning The foreign gene is transferred into a somatic cell, the nucleus of which is introduced into the cytoplasm of an enucleated oocyte to generate a transgenic clone.

20. Panel A, depicts the oocyte ready for enucleation. Panel B , depicts the nuclear material being removed from the cytoplast. The bottom portion of the figure shows the karyoplast transfer into the enucleated cytoplast. Panel C, depicts the insertion of the karyoplast transfer needle into the zona pellucida of the enucleated oocyte. Panel D, depicts the karyoplast after insertion into the zona prior to cell fusion into the cytoplast (Photographs courtesy of Jane H. Pryor and Charles R. Long, Texas A&M University).

21. “Dolly” 1996, first living offspring derived from a differentiated cell . (Ian Wilmut and Keith Campbell,1996)

22. Use of Transposons Transposons are short genomic DNA regions which are replicated and randomly integrated into the same genome. The number of a given transposon is thus increasing until the cell blocks this phenomenon to protect itself from a degradation of its genes. Foreign genes can be introduced into transposons in vitro. The recombinant transposons may then be microinjected into one day old embryos.

23. The foreign gene becomes integrated into the embryos with a yield of about 1%. All the transgenic insects are being generated by using transposons as vectors. Transposons also proved to be efficient to generate transgenic fish, chicken and mammals (Ding et al. 2005). Transposons are efficient tools but they can harbour no more than 2–3 kb of foreign DNA.

24. Efficient Generation of Transgenic Cattle U sing the DNA Transposon Here, efficiently generated transgenic cattle using two transposon systems ( Sleeping Beauty and Piggybac ). Sleeping Beauty is preferred for insertion into “TA” sites in the host genome, while Piggybac is preferred for insertions into “TTAA” sites Blastocysts derived from microinjection of DNA transposons were selected and transferred into recipient cows. Nine transgenic cattle have been generated and grown-up to date without any health issues except two. Some of them expressed strong fluorescence and the transgene in the oocytes from a superovulating one were detected by PCR and sequencing . (Yum et. al., 2017)

25. Birth of a transgenic ( tg ) cow with the YFP gene via Sleeping Beauty (SB) and its analysis. ( a ) After 60 days of embryo transfer, pregnancy was confirmed by ultrasonography. The calf was delivered without assistant ( b ) and grew to 5-months ( c ) and 16 months ( d ) old without any health issue. ( e ) When ultraviolet light was exposed to nose of tg cattle, YFP expression was found (arrow). To determine YFP expression in primary skin and endometrial cells, the cells were cultured and captured by confocal image equipment (( f-1 ) skin cells from a wild type, ( f-2 ) skin cells from a tg cattle, ( f-3 ) endometrial cells from a tg cattle , upper: brightness, lower: fluorescence). The primary skin cells from tg or non- tg were reprogrammed and developed into blastocysts ( f-4 ) blastocysts from skin cells of non- tg cattle, ( f-5 ) blastocysts from skin cells of the tg cattle; upper: brightness, lower: fluorescence ). The tg integration was confirm by PCR ( g ) and sequencing ( h ). (Yum et. al., 2017)

26. Birth of a transgenic ( tg ) cattle with the rox -GFP- rox -RFP gene via Piggybac (PB) and its analysis ( a ) After 45 days of embryo transfer, pregnancy was confirmed by ultrasonography. ( b ) The calf was delivered without assistant. ( c ) When ultraviolet light was exposed to nose of tg cattle, GFP expression was strongly observed. And the tg cattle grew up to 12 months old without any healthy issue ( d ). To determine GFP or RFP expression in a piece of tissue or primary skin cells via recombination, the tissue and cells were cultured and transfected with Dre recombinase mRNA by nucleofection (( e ) a piece of tissue from tg cattlebrightness , ( e` ) before Dre recombinase transfection (GFP), ( e`` ) after Dre recombinase transfection (RFP )). The primary skin cells from the tg cattle were isolated, cultured and transfected with Dre recombinase mRNA. Before transfection, only GFP expression was observed, RFP expression were observed via GFP gene excision by recombination (( f – f`` ) before transfection brightness, fluorescence, and merged, respectively; ( g – g`` ) after transfection brightness, fluorescence, and merged, respectively). The transgene integration and recombination were confirmed by genomic DNA PCR (( h ) (Yum et. al., 2017)

27. Sperm Mediated Gene Transfer The sperm cells have the capacity to bind naked DNA or bound to vesicles like liposomes ( Lavitrano et al., 1989; Chang et al., 2002 ). These sperm cells are in turn used for introducing exogenous DNA into oocytes either through invitro fertilization or artificial insemination. Sperandio et al. (1996) successfully carried out the sperm mediated gene transfer in cattle .

28. Electroporation This technique was developed by Puchalski and Fahl (1992). In this technique, cells are exposed to electric field which causes the membranes to become polarized and a potential develops across the membrane thereby breaking at localized areas . The cell becomes permeable to exogenous molecule. The method has a greater efficiency either alone or in combination with other.

29. Chemical technique This technique utilizes the chemical mediated uptake of DNA or gene fragment by the host cell. The transfection is carried out effectively by using chemicals like calcium phosphate or diethyl amino ethyl dextran .

30. RNA Interference In this method, small interference RNAs ( siRNAs ), which are 20-25 nucleotides long, bind to their complementary sequences on target in mRNAs and shut down the expression of genes and there by the production of protein is stopped . This RNA could be used for either transient or stable gene repression or knock down of specific target genes .

31. Some Examples of T ransgenic A nimals

32. Transgenic Fish Super fish Growth hormone gene inserted into fertilized egg. Increased growth and size. Transgenic fish grows about 10-11 time faster than normal fish . Glo fish Genetically modified zebra fish. Produced by integrating a fluorescent protein gene from jelly fish into embryo of fish.

33. 27 Super fish Glo fish

34. Transgenic Mouse Onco mouse Mouse model to study cancer. Made by inserting activated oncogenes . Alzheimer’s mouse In the brain of Alzheimer’s patients, dead nerve cells are entangled in a protein called amyloid. Mouse made by introducing amyloid precursor gene into fertilized egg of mice.

35. Transgenic Pig Enviro pig Pigs have trouble fully digesting a compound known as phytate in many cereal grains used to feed them. Transgenic pigs are created by introducing phytase gene of Ecoli . Phytase enzyme is thus produced in the salivary gland of pig. It degrades indigestible phytase with the release of phosphate that is readily digested by pigs . Pig for organ transplant Pigs with human genes, in order to decrease the chance of organ rejection by human body.

36. Transgenic Sheep For good quality wool.

37. Transgenic Monkey “ANDI” was the first transgenic monkey, born in 2000. ANDI proves that transgenic primates can be created and can express a foreign gene delivered into their genome.

38. Transgenic Cow “ROSIE” was the first transgenic cow , born in 1997. Produced human protein enriched milk at 2.4g/ lt contains human gene Alpha lactalbumin .

39. Importance of Transgenic Animals Industrial importance – Toxicity sensitive transgenic animals to test chemicals. Spider silk in milk of goat. Medical importance – Disease model. Xenotransplantation. Agriculture importance – Disease resistant animals. For improving quality & quantity of milk, meat, eggs & wool production.

40. Applications I n medical research, transgenic animals are used to identify the functions of specific factors in complex homeostatic systems . In toxicology: as responsive test animals (detection of toxicants ); In mammalian developmental genetics ; In molecular biology: the analysis of the regulation of gene expression makes use of the evaluation of a specific genetic change at the level of the whole animal.

41. In the pharmaceutical industry, targeted production of pharmaceutical proteins, drug production and product efficacy testing ; In biotechnology: as producers of specific proteins ; Genetically engineered hormones to increase milk yield, meat production . Developing animals specially created for use in Xenografting

42. Limitations of Transgenesis The transgenic technology even though has tremendous applications in livestock improvement programmes , still it has lots of limitations: Insertional mutations resulting in alteration of important biological processes. Unregulated gene expression resulting in improper expression of gene products. Possibility of side effects in transgenic animals like arthritis, dermatitis and cancer etc. Integration of exogenous DNA sequence in Y chromosome resulting in transmission only to males.

43. Issues related to Transgenic Technology There may be health risks associated with transgenics . There may be long term effects on the environment when transgenic animals are released into the field . Abnormalities suffered are more. Reduced fertility. Respiratory and circulatory problems. Weak immune system.

44. Ethical Concerns pertaining to Biotechnology Introduction of a transgene from one species into another species violates the “integrity of species”. Biotechnology may pose unforeseen risks to the environment, including risk to biodiversity. Transfer of human genes into animals ( and vice – versa ) dilutes the concept of “humanness ”. When animals are used for the production of pharmaceutical proteins, they are virtually reduced to the status of a “factory”. Use of animals in biotechnology causes great suffering to them. Biotechnology is disrespectful to living beings , and only exploits them for the benefit of human beings.

45. How the Ethical Issues can be Overcome: PATENTS …..! Avoid unnecessery repetition of experiments. Use immunochemical system to replace bioassays for detecting bacterial toxins. The scientists should not view animals as mere machines. India enacted an animal law as early as 1960's called the `Prevention of Cruelty to Animals Act' amended in 1982 which provided for the prevention of cruelty to animals in general. It also provides that the Animal Welfare Board constitute a Committee for the Control and Supervision of Experiments on Animals.

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