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Dr. Azhar Chishti Department of Medical Biochemistry Dr. Azhar Chishti.

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

2 LECTURE OUTLINES Dr. Azhar Chishti 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

3 Dr. Azhar Chishti 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.

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

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. Azhar Chishti

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: a. non-mutated protein, b. expression of a dominant-negative form of a protein, c. expression of a fluorescent-tagged protein. Dr. Azhar Chishti

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. Azhar Chishti

10 Microinjection equipment

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. Azhar Chishti

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

15 opsin promoter  globin MAR lacZ non-TgTg

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

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

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

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20 APP Ab 6E10 Non-Tg Tg CRND6 Tg CRND8 Tg APP kDa 12 kDa APP CTF C99 and A  Tg CRND Days Ab 6E kDa AA PS1 Ab NT-1kDa PS1 NTF  -stubs APP Protein expression from APP and PS1 transgenes

21 Age (weeks) n A  40 ng/g A  42 ng/g ± 3 54 ± 7 79 ± ± ± ± 4 55 ± 7 56 ± ± ± ± ± ± ± ± 0.6 A  42/40 Amyloid peptide in aging TgCRND8 mice

22 TgCRND8 Brain Pathology

23 Morris Water Maze Test “Swim path” Hidden platform

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

25 IAPP vaccine A  42 vaccine What effect on AD neuropathology? A  Immunization Causes a 50% Reduction Neuropathology of TgCRND8 Mice at Age of 25 Weeks

26 A  -immunization prevents/stops memory deficit in the TgCRND8 AD mouse Normal mouse AD mouse, Aß42AD mouse, IAPP ctl Normal mouse AD mouse, Aß42 AD mouse, IAPP ctl Before Training After Training

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

28 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. Azhar Chishti

29 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.exons 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. Azhar Chishti

30 Knockout mice Dr. Azhar Chishti. Figure 1: Gene targeting for knockout mice

31 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. Azhar Chishti

32 Conditional Knockout mice Dr. Azhar Chishti. Figure 1: Gene targeting for knockout mice Figure 1: Gene targeting for knockout mice

33 Conditional Knockout mice Cre recombinase 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. Azhar Chishti

34 Presenilins N C ER Cytoplasm

35 Superimposed transgene None TgPS1(M146L)1 TgPS2(N141I)1032 TgPS2(M239V)1379 TgAPP 6209 PS1 deficient mice

36 33 d. 33d 62d PS1 genes accelerate amyloid deposition in TgCRND8 mice minus PS1(M146L+L286V)PS1(L286V)

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

38 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. Azhar Chishti

39 References Lippincott, Illustrated review of Biochemistry, 4th edition Transgenic and Knockout Mouse – Approaches (Website) html Chishti MA et al.,. Early-onset amyloid deposition and cognitive deficits in transgenic mice expressing a double mutant form of amyloid precursor protein Journal of Biological Chemistry. 276: Dr. Azhar Chishti

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


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