L5-L6 - Transgenic animals and its applications.pptx

L5-L6 - Transgenic animals and its applications

1. Transgenic animals V. Rajanbabu BTB 404 Molecular Pharming (1+1) B. Tech. (Biotech.), CPMB&B, TNAU, Coimbatore Lecture 5 1

2. Transgenic animals: Definition An animal that gains new genetic information from the addition of foreign DNA is described as Transgenic while the introduced DNA is called the transgene Transgenics are genetically modified organisms with DNA from another source inserted into their genome Animal has one or more foreign genes inserted into its chromosomes A transgenic animal is animal whose chromosomes have been changed to carry the genes of other organisms 2

3. Transgenesis process of introducing foreign or exogenous DNA into an animal’s genome Transgene DNA introduced Mice Cows Fish Birds Sheep Goats Definitions 3

4. Why Transgenesis in animals? Improve genetic Features of domesticated Animals Authentic post-translational modifications Pharming : Using farm animals for production of human pharmaceuticals Study the genes regulation, development of animals 4

5. First transgenic animal 5

6. Knockout Mouse : A transgenic mouse in which the normal gene is missing or engineered so that is not transcribed or translated. “Knocks out” that gene. Knockin Mouse : A transgenic mouse in which the engineered “ transgene ” is subtly manipulated to: (A) alter the function of the gene (e.g., replace one amino acid with another in a site to determine if that site is essential for the protein’s function); (B) change transcription rate to overproduce or underproduce the gene product; or (C) create a fluorescent gene product to map its distribution in tissue. Conditional Knockout ( Knockin ) Mouse : A transgenic mouse in which the transgene is knocked out (or in) in specific tissues, at a specific developmental stage, or in response to an exogenous substance (e.g., an antibiotic). Transgenic mouse 6

7. Knock-out technology allows for the specific loss of a gene in mice Allows for the function of the gene to be deduced from the defects seen in the mice can be used to mimick some disease Unlike traditional transgenics the trangene is targeted to a specific site in the DNA of the mouse Knock-out 7

8. http://cba.musc.edu/SC_COBRE/CORE-B/Resources-B.htm Mouse Knock-outs require embryonic stem (ES) cells These are derived from the inner cell mass (ICM) of a blastocyst (the ICM is what will become the fetus) ES cells are pluripotent meaning they can become all the different cell types found in an adult 8

9. http://tasq.uq.edu.au/blasto.html Blastocyst Injection Blastocyst ES cells 9

10. Pronucleus For a short time after fertilization, the male pronucleus and female pronucleus exist separately Female pronucleus ; In the maturing of the ovum preparatory to impregnation, a part of the germinal vesicle becomes converted into a number of small vesicles, which aggregate themselves into a single clear nucleus which travels towards the center of the egg and is called the female pronucleus Male pronucleus ; In impregnation, the spermatozon which enters the egg soon loses its tail, while the head forms a nucleus, called the male pronucleus , which gradually travels towards the female pronucleus and eventually fuses with it, forming the first segmentation nucleus. The male pronucleus is larger than the female’s and can be seen fairly easily under a light microscope 10

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12. 12 Transgenesis process Plasmids carrying the gene of interest are injected into the germinal vesicle (nucleus) of the oocyte or into the pronucleus (before uniting with the gamete) of the fertilized egg. The egg is implanted into a pseudopregnant mouse After birth, the recipient mouse can be examined to see whether it has gained the foreign DNA and if so whether it is expressed. As a result; multiple copies of transgenes are integrated at random locations in the genome of the transgenic individuals. The transgenes in many transgenic individuals are also transmitted through the germline to subsequent generations.

13. How to Get the Transgene Inserted Chemical method ( Transfection ) : Lipofectamine Physical method ( Transformation ): Microinjection Biological method Bacteria ( Bactofection )- the gene will be transferred as a plasmid Virus ( Transduction )- the transferred gene represents part of the viral genome 13

14. Chemical transfection (1/2) DNA will be taken from the surroundings when the DNA is presented as a synthetic complex either as; a complex with overall positive charge, allowing it to interact with negatively charge cell membrane and promote uptake by endocytosis as lipophilic complex that fuses with the cell membrane and deposits the transgene directly into the cytoplasm. 14

15. Chemical Transfection Techniques (2/2) Calcium phosphate method; involves the formation of a fine DNA/calcium phosphate co-precipitate which first settles on the cells and then internalized by endocytosis . Polyplexes ; this is more efficient than that with calcium as it gives more uniform particle size. Polyplexes are a polycationic compounds that form soluble complexes ( polyplexes ) through spontaneous electrostatic interaction with DNA. liposomes and lipoplexes : packaging the DNA inside a fusogenic phospholipid vesicle which interacts with the target cell membrane and facilitate DNA uptake 15

16. Physical transfection Naked DNA is deposited directly into the cell by exploiting a physical force. This includes; Microinjection Particle bombardment Ultrasound Electroporation Which ever method used, the result is called transformation which is a change in the recipient cell ’ s genome caused by the acquired transgene . 16

17. DNA microinjection (1/2) Direct transfer of DNA into the cell without a carrier Few cells at a time This technique is used mainly for large cells such as oocytes , eggs and the cells of early embryos. The DNA can be directly injected into tissues, such as skin, muscle or internal organs or it can be injected into the blood. 17

18. DNA microinjection (2/2) Remarkably efficient . 60- 66%% of the embryos survive 25-30% of the embryos transferred to the oviduct survive to birth 25% of pups are transgenic (transgenic founders). Thus, from 1000 inoculated fertile eggs, 30-50 (3-5%) transgenic pups are produced. Disadvantage : The injected DNA gets incorporated at random sites within the genome and often multiple copies are incorporated at one site, therefore, not all the transgenic animals will have the desired traits. 18

19. Particle bombardment Another direct delivery method initially developed for the transformation of plants. Coating small metal particles with DNA and accelerating them into target tissues using a powerful force such as the blast of high pressure gas or an electric discharge through a water droplet. In animals, this method is used for tissues such as skin cells in vivo rather than cultured cells. 19

20. Ultrasound Involves the exposure of cells to a rapidly oscillating probe such as the tip of sonicator . Cells or a particular tissue results in the formation and collapse of bubbles in the liquid, including the cell membrane, a process known as cavitation . Cavities allows the DNA to cross the membrane into the cytoplasm. in vivo or in vitro as the plasmid DNA is left structurally intact. DNA will be injected and then the ultrasound will be applied. 20

21. Electroporation Physical transfection technique involves creating transient nano -meter size pores in the cell membrane by exposing cells to a brief pulse of electricity. The most critical parameter is the intensity and duration of electrical pulse. Used for in vivo gene transfer: Near surface tissue such as skin, muscle and certain tumors or even internal tissues such as liver. Direct application of electrodes to the skin following shaving and mild abrasion 21

22. Biological method 22

23. Bacterial vectors for gene transfer (1/3) Agrobacterium tumefaciens and its close relatives have been used for 20 years to generate transgenic plants. A. tumefaciens has been reported to transfer DNA to human cells 23

24. How does the bacterial transfer of DNA happen? (2/3) The bacteria invades the host animal cells and undergo lysis within them releasing their plasmid DNA. Example Salmonella species (lysis occurs in the phagocytic vesicle), Listeria monocytogenes and Shigella flexneri (lysis occurs after they escape from the vesicle). 24

25. The plasmid DNA then finds its way to the nucleus where it gets incorporated with the cell ’ s genome and gets expressed. Contrary to bacterial strains in the previous slide, A. tumefaciens does not invade the cell, instead, it attaches itself to the outside surface followed by conjugation. Bacterial vectors for gene transfer (3/3) 25

26. Viruses That are used as gene transfer vectors (1/3) Virus particles have a natural ability to adsorb to the surface of the cells and gain entry. Transgenes are incorporated into viral vectors either by addition to the whole viral genome or by replacing one or more viral genes. This can be done by ligation or by homologous recombination. 26

27. If the transgene replaces a none essential gene the vector is described as helper-independent If it replaces an indispensable gene, then this vector will be helper dependent . Generally all viral coding sequences has been deleted, and such vectors are described as fully deleted or gutted or gutless vectors. These vectors contain just the cis -acting elements required for packaging and for genome replication. Viruses That are used as gene transfer vectors (2/3) 27

28. Advantages of virus vectors (3/3) high capacity for foreign DNA because no viral gene products are made, the vector has no intrinsic cytotoxic effects. 28

29. 3 Pig clones, born in 2002, died of heart attacks due to “adult clone sudden death syndrome” within days of each other by the time they were 6 months old . Dolly had a weight problem, telomeres 20% shorter than normal, she suffered from arthritis, and finally lung cancer due to an infection for which she was finally euthanized at age 6yrs . The success rate ranges from 1 to 3% this contrasts to in vitro fertilization which has a success rate of 50 to 20% Problems with Cloning 29

30. Animals as biofactories : Production of products of therapeutic value 30 BTB 404 Molecular Pharming (1+1) B. Tech. (Biotech.), CPMB&B, TNAU, Coimbatore Lecture 6

31. recombinant protein production in animals/animal cells Construct design for high-level transgene expression The use of a strong and constitutive promoter Inclusion of an intron Inclusion of a polyadenylation signal Removal of unnecessary untranslated sequence Optimization of the transgene for translational efficiency Incorporation of a targeting signal 31

32. Cell-free expression High throughput applications Eg . bacterial, wheat germ and reticulocyte lysates Yields of up to 6 mg/ml Optimal concentrations have to be identified and carefully controlled 32

33. What Has Been Cloned So Far? Somatic Cell Nuclear Transfer Sheep, Goat, Mouse, Rabbit, Cattle (domestic & wild), Pig, Horse, Mule, Dog, Cat (domestic & wild), Deer Embryo Splitting (Twinning) Sheep, Cattle, Primate (Rhesus) 33

34. http://nolswf.bbc.net.uk/science/genes/gene_safari/pharm/a_pharming.shtml 1997, Tracy the sheep, the first transgenic animal to produce a recombinant protein drug in her milk alpha -1-antitrypsin (AAT) treatment for emphysema & cystic fibrosis Created by PPL Therapeutics & The Roslin Institute recombinant protein drug in sheep milk 34

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36. Webster and Peter Nexia Biotechnologies transfered the silk gene from Orb spiders into goats The resulting male goats were used to sire silk-producing female goats Each goat produces several grams of silk protein in her milk The silk is extracted, dried to a white powder, and spun into fibers The fibers are stronger and more flexible than steel Transgenic male kids carrying silk gene 36

37. GTC Biotherapeutics has received approval to sell human anti-thrombin ( ATryn ) purified from goat’s milk in Europe Technology is not restricted to cows, goats, & sheep There is interest in using rabbits since housing costs are significantly less & generation time is faster Chickens which produce recombinant drugs in their eggs have been produced by The Roslin Institute ATryn 37

38. First transgenic cow Rosie – 1997 Human Alfa lactalbumin enriched milk at 2.4g/ lt 38

39. Genetically modified cows produce 'human' milk "human" milk in a bid to make cows' milk more nutritious human protein lysozyme , lactoferrin , alpha- lactalbumin 39

40. Herman – The bull   First transgenic mammal (1990)   human gene coding for lactoferrin 40

41. featherless Chickens By inserting  human protein encoding genes into the chicken's genome, the eggs will be enriched Transgenic featherless chickens- keeps cold and grow comfortable in hotter climates 41

42. EnviroPig TM Transgenic pigs express phytase in their salivary glands Phytic acid in the pig meal is degraded releasing phosphorus The phosphorus is absorbed by the pig Normally the phytic acid/phosphorus complex passes through the pig and is excreted as waste Pig waste is a major pollutant & can cause eutrophication of lakes & streams http://www.nature.com/news/2005/050221/images/nbt0305-283-I1.jpg 42

43. Transgenic Fish Tilapia Salmon/trout Catfish Can grow up to 6 times faster than wildtype fish Most have extra copies of growth hormone (GH) gene Transgenic Wildtype http://www.nature.com/nbt/journal/v19/n6/images/nbt0601_500a_I1.jpg 43

44. Lecture Schedule - Midsem Concept of biopharming Use of microbes, plants and animals for synthesis of therapeutic and pharmaceutical compounds Overview of techniques used in biopharming Nature of microbes used as biofactories : Microbial production of insulin, recombinant proteins of therapeutic or industrial value. Transgenic animals Animals as biofactories : Production of products of therapeutic value Plants as biofactories : Transgenic plants for production of heterologous proteins Plants as delivery systems of vaccines and plantibodies