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PSY355 lectures.pdf

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Robert Gerlai

PSY355Lecture - 20NOV 2013 Notebook: PSY355 Crea ted: 11/20/2013 12:06 PM Upda ted: 11/20/2013 1:56 PM 23. don't know if phenotypical change from knockout is due to change in function of the mouse's brain 129 easy tobreedinvitroandsurvive well, easy tomaintain pure genetic background seems like a easy solution but difficult because it is hard to get B6 type embryonic stem cells and are hard to breed/maintain 24. backcrossing:cross 129type withb6--> cross F1generationagainwithB6 (desired genetic background) repeatedly with many generations of back crosses, area around target locus becomes smallerandsmallerinterms of 129 type alleles the shorter the distance on a chromoso me, the less likely itwill be that a recombination occurs speedcongenics:speedupprocess of replacing alleles, helps tospeedupback crossing process by helping you get closer and closer to target alleles 25. allows you to restrict gene targeting events to particular cell types its often very difficult to tell the effect of the knockout, or see if observable effects are even as a result of the knockout or not CRE: is a bacterial enzyme that doesn't exist in eukaryotic organisms like us, evolutionary ancient enzyme recognizes particular short 12 nucleotide long sequences that don't exist in humans enzyme when finds sequence LoxP site , binds to it, if finds two, will physically cut out whatever is in between these two sites (unique restriction endonuclease) promoter regulates gene expression, CamKII promoter turns on Cam-kinase gene late, after mouse brain development is almost complete if you create a genetic construct to add LoxP around targeted gene knockout only happens in the forebra ni, because CamKII only works in the forebrain, CRE is not expressed anywhere else 28. creating mice where gene expression is inducible requires two transgenic lines one in which CamKII promoter, tTA which encodes a protein that is a transcription factor second tetO attached to gene of interest tTA can be expressed and if it binds to tetO gene will be expressed, protein production occurs if youinject anantibiotic tomouse with these genes, then it blocks the binding of tTA protein so you can turn off gene expression doxycycline cansignificantly affect the fetus, giventomotherbefore she is even pregnant to block gene X expression in adulthood of mutated mouse 29. rtTA disfunctional protein is unable to reduce gene expression of gene X, only becomes functional when it finds an antibio it to bind to which allows it to bind to tetO to induce gene expression don't have to give to mother, only give antibiotic to adult mouse after brain development 32. conditional GENE EXPRESSION (not a knock out as title says in ppt) 33. PSY355Lecture - 6NOV 2013 Notebook: PSY355 Crea ted: 11/6/2013 11:41 AM Upda ted: 11/6/2013 2:19 PM 1. 2. 3. 4. 5. 6. technology is powerful because saves a lot of time, effort, money since you can target genes using technique in petri dish in less than a day vs. months of screening mice null mutant mouse created b/c disrupts gene so that there is no protein production = not normal function targeting vectorcontains a sequence thatis almostidentical togene in question, only region which vector is homologous is the gene you are interestedin 7. sequence of gene of interest, boxes represent transcribed areas of the gene (1,2,3,4 are coding regions for amino acid sequence of the proteins) neostands forneomycinresistance, like a little gene thattranslates a protein, actually a bacterial gene tk, sequence encodes a protein that once expressed phosphorylates targeted proteins (is a kinase) creates a toxin once performing this action 8. howa sequence looks like whenitis cut, doesn't cut ina straightline, creates stickyends 9. presence of neomycin you can test by treating embryonic stem cell with G418 10. -12. replacement occurs only in places where there is homology neomycin is incl. tk (toxic part) won't be there treating mice with toxic drug, only those with true homologous replacements will survive (i.e. those without the tk incl.) three scenarios 1. if targeting vector does not incorporate, then neomycin gene is absent, ES cell not resistant to drug g418 2. random insertion of vector doesn't replace endogenous gene tk doesn't go in because not homologu os to endogenous gene, reason why tk gene is at end of construct and used as seconday rto show incorporation of neomycin 11. 12. 13. two methods: 1. blastocyst injection, 2. morula aggregation 2. clump of embryonic stem cells from host sandwiching clump of ES cells 14. two kinds of mice can come from pseudo-pregnant mother only host derived mouse (no ES cell contribution to its body) 10% of mice will have embryonic stem cells represented embryonic stem cell comes from different colored mouse genome, reason why mice with embryonic stem cells have patchy diff. coloured fur) want testes/ovaries to develop from embryonic stem cells so that mouse can pass on genes of mutation to its offspring 15. viability of mutant offspring can be problematic, 25% rate of having homozygous mutant can be greatly reduced from mice who don't go through all the correct stages of embryonic development and die before birth or mice who are born but are just not viable 16. lack of functional gene product results in non mutant mouse b/c other genes compensate for the function of the gene that was knocked out 17. veryhardtoassignfunctiontotargeting gene basedonthis system very difficult question to answer using this technology 18. thought experiment 19. gene targeting allows us to understand how a system is organized 20. can minimize compensatory effects inducible systems: allow embryonic development to happen normally, introduce altered gene in later stage of development 21. false negative: don't find a difference when there is one present if variability is high between mutants and non mutants it can be very difficult to detect a difference PSY355Lecture - 30OCT2013 Notebook: PSY355 Crea ted: 10/30/2013 11:02 AM Upda ted: 11/13/2013 11:36 AM 1: Lecture 7: Transgenic + Knockout Mice many diff. types of reverse genetics techniques 2: transgenic overexpressors:foreign gene put into genome of mouse in large number of copies, looking at overexpressing a
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