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Transgenic Animals

Transgenic Animals. Dr. Azhar Chishti Department of Medical Biochemistry. LECTURE OUTLINES. Transgenic animals Overview of transgenic mice How to create transgenic animals Transfer of DNA into eukaryotic cells Confirming mutation in germ cells Overview of knockout mice

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Transgenic Animals

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  1. Transgenic Animals Dr. Azhar Chishti Department of Medical Biochemistry Dr. Azhar Chishti

  2. LECTURE OUTLINES • Transgenic animals • Overview of transgenic mice • How to create transgenic animals • Transfer of DNA into eukaryotic cells • Confirming mutation in germ cells • Overview of knockout mice • How to make gene targeted knockout mice • Making ES cell containing knockout mutants • Forming chimeras • generating homozygous knockout mice • Use of transgenic & knockout mice to study human genetic diseases • Use of transgenics in gene therapy e.g. SCID Dr. Azhar Chishti

  3. Transgenic animals can be produced by injecting a cloned gene into the fertilized egg. • A giant mouse called "Supermouse“ • Transgenic goats and cows can produce human hormones in their milk. • Sometimes, rather than introducing a functional gene into a transgenic mouse, a mutant gene is used to replace the normal copies of that gene in the cells of the mouse. • This can be used to produce a colony of "knockout mice" that are deficient in a particular enzyme. • Transgenic animals can serve as models for the study of a corresponding human disease. For example, transgenic mice of dystrophin gene serve as animal models for the study of muscular dystrophy. Transgenic Animals Dr. Azhar Chishti

  4. Transgenic Animals 1. Transgenic mice 2. Knockin and knockout mice 3. Scid mice Dr. Azhar Chishti

  5. Transgenic mice • Developments in molecular biology and stem cell biology over the last 20 years have allowed researchers: • To create custom-made mice through gene targeting in mouse embryonic stem (ES) cells. • Site-directed mutagenesis in embryonic stem cells and the phenotypic characterization of the corresponding knockout and/or knockin mouse • allows researchers to study gene function as it relates to the entire organism • Now, certain diseases that normally do not present in mice, such as cystic fibrosis and Alzheimer's disease can be induced by manipulating the mouse genome and environment. Dr. Azhar Chishti

  6. Transgenic mice • Transgenesis is the introduction of DNA from one species into the genome of another species. • Transgenic mouse is generally refers to any mouse whose genome contains an inserted piece of DNA, originating from the mouse genome or from the genome of another species. • To study gene function in a mouse is exogenous expression of a protein in some or all tissues. Dr. Azhar Chishti

  7. Transgenic mice (cont.) • The piece of DNA includes: a. the structural gene of interest b. a strong mouse gene promoter c. Enhancer to allow the gene to be expressed d. Vector DNA to enable the transgene to be inserted into the mouse genome. Dr. AzharChishti

  8. Transgenic mice (cont.) • Successful insertion of DNA results in the expression of the transgene in addition to the wild type, basal (endogenous) protein levels in the mouse. • Depending on the goal of the experiment, the transgenic mouse will exhibit over-expression: • non-mutated protein, • expression of a dominant-negative form of a protein, • expression of a fluorescent-tagged protein. Dr. AzharChishti

  9. Transgenic mice (cont.) • To generate a standard transgenic mice include bacterial or viral vector containing the transgene and any desired markers are injected into a fertilized mouse egg. • The DNA usually integrates into one or more loci during the first few cell divisions of preimplantation development. • The number of copies of the transgenic fragment can vary from one to several hundred and the transgenic founder mice are mosaic for the presence of the transgene. • Founders are very likely to have germ cells with the integrated transgene, and therefore will be able to vertically transmit the integrated gene, and all cells of the progeny transgenic mouse contain the transgene. Dr. AzharChishti

  10. Dr. Azhar Chishti

  11. Transgenic mice (cont.) • This method is relatively quick, but includes the risk: a. DNA may insert itself into a critical locus, causing an unexpected, detrimental genetic mutation. b. Transgene may insert into a locus that is subject to gene silencing. c. If the protein being expressed from the transgene causes toxicity, excessive overexpression from multiple insertions can be lethal to some tissues or even to the entire mouse . • several independent lines mice containing the same transgene must be created and studied to ensure that any resulting phenotype is not due to toxic gene-dosing or to the mutations created at the site of transgene insertion. Dr. AzharChishti

  12. Why mice and why transgenics? • Mice are a common “platform” shared with other biomedical researchers • Mice can recapitulate key pathologies observed in human disease • Trangenes allow access to all forms of a protein - better than toxicological phenocopy of disease. • ENU no good - need humanized genes

  13. The retina as a target for spontaneous prion formation • simple architecture • photoreceptors replicate prions. • prions can transit between the retina and the CNS. • prions formed in the retina can be amplified in the CNS, travel to the periphery. Brain

  14. * To brain Insult Optic nerve Retinal photoreceptors as a target for de novo prion formation Lens

  15. lacZ e globin MAR opsin promoter non-Tg Tg

  16. PrP E200K PrP F-CJD mutations E200K V210I PrP Retinal transgenes lacZ e globin MAR opsin promoter

  17. Asymmetric photoreceptor cell degeneration in opsin/PrP mice Superior Hemisphere (SH) Inferior Hemisphere (IH)

  18. ATG TAG APP695 KM670/671NL V717F 42 kb Hamster PrP cosmid • Cosmid injected into C57B6/C3H oocytes • Resultant line is denoted “TgCRND8” • Maintained on C57B6/C3H outbred or 129SvEv congenic

  19. Tg CRND6 Tg APP6209 Tg CRND8 Non-Tg 1 2 3 4 5 36 PS1 NTF 30 kDa Ab NT-1 Protein expression from APP and PS1 transgenes Tg CRND 8 60 120 180 240 300 Days C99 and Ab b-stubs 6 APP Ab 98 4 kDa Ab 6E10 64 50 APP 36 30 PS1 16 12 kDa APP CTF kDa Ab 6E10

  20. n Ab42 ng/g Ab40 ng/g Ab42/40 6 7 7 5 5 38 ± 3 54 ± 7 79 ± 30 409 ± 245 21780 ± 6600 54 ± 4 55 ± 7 56 ± 3 101 ± 76 10584 ±1495 0.7 ± 0.02 1.1 ± 0.1 1.5 ± 0.5 5.2 ± 1.5 2.0 ± 0.6 4 6 8 10 25 Amyloid peptide in aging TgCRND8 mice Age (weeks)

  21. TgCRND8 Brain Pathology

  22. Morris Water Maze Test Hidden platform “Swim path”

  23. Learning acquisition in 11 week old TgCRND8 mice TgCRND8 Latency (seconds) Day 1 Day2 Day 3 Day 4 Day 5 Session

  24. IAPP vaccine Ab42 vaccine Ab Immunization Causes a 50% Reduction Neuropathology of TgCRND8 Mice at Age of 25 Weeks What effect on AD neuropathology?

  25. Normal mouse AD mouse, IAPP ctl AD mouse, Aß42 After Training Normal mouse AD mouse, IAPP ctl AD mouse, Aß42 Ab-immunization prevents/stops memory deficit in the TgCRND8 AD mouse Before Training

  26. Creating lab models of AD And the Oscar goes to… TgCRND8

  27. Knockin mice • A knockin mouse is generated by targeted insertion of the transgene at a selected locus. • Site-specific knockins result in a more consistent level of expression of the transgene from generation to generation because it is known that the overexpression cassette is present as a single copy. • The targeted transgene is not interfering with a critical locus and the resulting phenotype is due to the exogenous expression of the protein. • The generation of a knockin mouse does avoid many of the problems of a traditional transgenic mouse, this procedure requires more time to assemble the vector and to identify ES cells that have undergone homologous recombination. Dr. AzharChishti

  28. Knockout mice • Homologous recombination allows to completely remove one or more exons from a gene which results in the production of a mutated or truncated protein or, more often, no protein at all. • knockout mice are generated to remove protein information by elimination of a gene or the deletion of a functional domain of the protein. • This can be achieved through random mutation using chemical mutagenesis or a gene trap approach, or through gene targeting to generate a knockout mouse. Dr. AzharChishti

  29. . Figure 1: Gene targeting for knockout mice Knockout mice Dr. AzharChishti

  30. Conditional Knockout mice • Many genes that participate in interesting genetic pathways are essential for either mouse development, viability or fertility. • Therefore, a traditional knockout of the gene can never lead to the establishment of a knockout mouse strain for analysis. • Conditional gene modification using Cre-lox technology allows the gene of interest to be knocked-out in only a subset of tissues or only at a particular time to avoid lethality. • This genetic dissection allows researchers to define gene function in development, physiology or behavior. Dr. AzharChishti

  31. . Figure 1: Gene targeting for knockout mice Figure 1: Gene targeting for knockout mice Conditional Knockout mice Dr. AzharChishti

  32. Conditional Knockout mice • Crerecombinase is isolated from the P1 bacteriophage, catalyzes recombination between two of its consensus DNA recognition sites. • These loxP sites are 34 base pairs in length, consisting of two 13bp palendromic sequences that flank a central sequence of 8bp which determines the directionality of the loxP site. • Two loxP sites are most often placed on either side of an essential, functional part of a gene so that recombination removes that functionality and knocks-out the gene. (See Figure 2) • LoxP sites placed on different chromosomes can be used to generate targeted translocations, though this recombination event occurs at a relatively low frequency compared to the highly-efficient intra-gene recombination Dr. AzharChishti

  33. Presenilins Cytoplasm N C ER

  34. PS1 deficient mice Superimposed transgene None TgPS1(M146L)1 TgPS2(N141I)1032 TgPS2(M239V)1379 TgAPP6209

  35. PS1 genes accelerate amyloid deposition in TgCRND8 mice 33 d. 33 d. 33d 62d 33d minus PS1(M146L+L286V) PS1(L286V)

  36. HUMAN MOUSE Amyloid deposition in AD transgenic MOUSE brain (92 days of age). Amyloid deposition inHUMAN Alzheimer Disease Brain (70 years of age). A Transgenic Mouse Model of Human Alzheimer’s Disease

  37. SCID mice • These are immunodeficient mice • Nude mice are T-cell deficient • Scid mice are both T & B cell deficient • They are use in Cancer studies • They are use for gene therapy Dr. AzharChishti

  38. References • Lippincott, Illustrated review of Biochemistry, 4th edition • Transgenic and Knockout Mouse – Approaches (Website) http://www.cellmigration.org/resource/komouse/komouse_approaches.shtml • Chishti MA et al.,. Early-onset amyloid deposition and cognitive deficits in transgenic mice expressing a double mutant form of amyloid precursor protein 695. 2001 Journal of Biological Chemistry. 276:21562-21570 Dr. Azhar Chishti

  39. How to Improve your memory and intelligence? Follow the leaders!!!

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