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Mouse Genetics Final Exam Review Why study vertebrates/ mice? Reverse Genetics • What is it? – Starting with a specific genotype and figuring out the phenotype • Why is it used in mice? – Reverse genetics is slow and expensive in mice – Can test a candidate gene’s effects on complex behaviors, organs, etc. Tools for forward genetics in mice 1. Transgenes 2. Knock-ins Transgenes • What are they? – Engineered pieces of DNA that have been inserted randomly into the mouse genome by integration • Why use transgenes? – Any situation where you want to add in a gene and not remove or replace anything. • • • • • Test sufficiency/ complementation Label cell types/ organs of interest Add toxins Promoter bashing Etc. What do you need to make a transgene? • A promoter- initiates transcription • Your transgene- include a stop codon if protein encoding (as opposed to RNAi) • An intron- stops mRNA from being seen as foreign by the cell • Poly(A)- stop translation How can you make the transgene construct? 1. Bacterial plasmid - pros: easy and cheap Cons: small (20 kb) can be subject to position effect need to separate from vector 2. BAC (bacterial artificial chromosome) -pros: bigger (100-150 kb)- therefore can include more regulatory elements -cons: can be subject to position effect 3. Lentivirus -pros: less susceptible to position effect (targets active genes) can be used for different species no pronuclear injection -cons: small can disrupt essential genes How do you introduce the construct and make the mouse? Using a plasmid or BAC: 1. Make the transgene cassette and separate if from the vector DNA 2. Inject into male pronucleus of fertilized eggs before the nuclei have fused 3. Transfer the transgenic eggs to a surrogate mouse 4. Pups are born- genotype them to determine if the transgene has integrated. 5. Mate the founders with WT mice to see if you get transmission of your transgene. How do you introduce the construct and make the mouse? Using a lentivirus: • Same strategy as before, just no nuclear injection. • Incubate your fertilized egg with the viral supernatant instead. What problems may you encounter? No expression or mis-expression of transgene. • Position effect- transgene inserted into a part of the genome not accessible by the appropriate transcriptional machinery. – Solution? Try using a lentivirus • Your construct may have lacked the necessary regulatory regions – Use a BAC to get more upstream regulatory sequence. Gene Targeting • What is it? – Replacement or modification of an endogenous gene with a sequence of interest • Why use it? – Knockouts – Replacements – Targeted mutations What do you need to make a gene targeting construct? Components you must have: – arms of homology to allow recombination into the endogenous locus- at least 4kb on one side and 1.5kb on the other – Neomycin (or other antibiotic) resistant marker with it’s own promotor (usually a ubiquitous promoter), start and stop codons, and poly(A) tail- to select your ES cells – Put Frt or LoxP sites around your antibiotic resistance marker so you can remove it later Other tools you can use in gene targeting constructs • IRES (internal ribosomal entry sites) elements- allow you to translate multiple proteins from the same mRNA (allows 7mG-cap independent translation) • Reporter Genes (fluorescent proteins, LacZ, etc)- can be used in two ways – Fusion proteins- marks where IN THE CELL your protein of interest is – Cell labeling- marks WHICH CELLS are expressing your gene of interest • Use of tau:GFP fusions to label axons • Toxins (such as diphtheria toxin)- to kill cells of choice • Lox/ Cre and Flp/ Frt systems to selectively remove portions of your construct • Inducible promoters (tetracycline regulatory systems)- to allow or inhibit expression of your targeting gene How to make a construct for gene targeting 1. Order a BAC clone spanning your region of interest. - This should be from the same strain. Why? - So arms of homology do not contain SNPs 2. Screen a genomic library 3. PCR your region of interest 4. Synthesize it How to make a “gene targeted” mouse 1. Add your construct into ES cells from male brown mice and electroporate. 2. Select with appropriate antibiotics to get only cells which have undergone homologous recombination. 3. Inject the ES cells into blastocysts from black mice. 4. Place blastocysts into recipient female. Chimeras are born! 5. Mate chimeras to wild-type black mice 6. Look for brown progeny. 7. Mate to Flp/ Cre containing mice to remove selection cassette What do your chimeras look like? What does this mean? • All black pups • Pups with variegated coats • All brown females • All brown males What problems might you encounter? • Aberrant expression of knock-in – Forgot to “Flip-out” your antibotic resistance gene – It has it’s own promotor and poly(A) tail, which could lead to increased transcription and stability of your knock-in construct Other tools in mouse genetics 1. In-situ hybridization (ISH) 2. Nuclear cloning In-situ hybridization • Create a probe complementary to your mRNA of interest. Label this probe to determine where your gene of interest is being expressed. Nuclear Cloning • Creating an animal from a single adult cell Notes about mice • Sex- presence of Y chromosome determines sex- XX= female, XY= male • Because females have two X chromosomes, the gene products must be equalized. – X-inactivation- one X chromosome is stochastically and irreversibly inactivated. • If you have a loss of function mutation on the X chromosome, here is what mutants will look like: – X(lof)/ X female= – X(lof)/ Y male=