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Coding region

Transcription start (mRNA start) (mRNA end) poly-adenylation. ATG: Translation start Translation stop. Gene structure. exon intron. Regulatory regions. Coding region. Mature mRNA. AAA. cap. 5’UTR 3’UTR polyA tail.

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Coding region

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  1. Transcription start (mRNA start) (mRNA end) poly-adenylation ATG: Translation start Translation stop Gene structure exon intron Regulatory regions Coding region Mature mRNA AAA cap 5’UTR 3’UTR polyA tail

  2. Transgenic gain-of-function (transgene injection in fertilized eggs) Gene of interest with it’s own regulatory elements Gene of interest Heterologous regulatory region cDNA from gene of interest Heterologous regulatory region

  3. BAC transgenics GFP recombineering ~ 200 kb Regulatory elements can many kb away from gene

  4. Gene inactivation (constitutive) by gene targeting in ES cells Simple targeting construct NEO Positive selection Replace critical exon with Neomycin resistance gene NEO Heterozygote +/- NEO Homozygote -/- NEO

  5. Targeted gene knock-in (knock-out) in ES cells Note insertion between transcription and translation start sites CRE Targeting construct NEO NEO Positive selection CRE TK DTa NEO NEO Negative selection Cre (or a reporter gene) is now driven by regulatory elements of gene of interest. Critical parts of gene of interest are removed so it is also a null allele. CRE NEO NEO

  6. Frt sites Gene of interest NEO NEO CRE ires-Cre Gene of interest after FLP recombinase action CRE Use of ires-Cre does NOT disrupt function of gene of interest

  7. Mouse with Cre expressed from gene of interest, e.g. Agrp-Cre a) Breed with mouse containing conditional allele of gene of interest to either inactivate (or activate) that conditional allele b) Inject a Cre-dependent virus (to achieve brain region specificity) DIO FLEX C -------------N ITR promoter gene of interest WPRE pA ITR backwards First recombination (inversion), intermediate stage N -------------C Second recombination (deletion), locks it into inverted position N -------------C ITR promoter gene of interest WPRE pA ITR backwards Double recombination results in expression of gene of interest

  8. NEO Conditional gene inactivation Neo may disrupt function = KO Targeted allele Removed with FLP recombinase Frt-NEO loxP sites Remove Frt-NEO With FLIP recombinase Breed to homozygosity and introduce Cre gene CRE Cell-specific expression of Cre recombinase (transgene or knock-in)

  9. Cre-mediated recombination wherever Cre expressed (loss of critical exon) CRE Cre-mediated recombination wherever & whenever Cre is expressed (loss of critical exon) CRE ERt Cell-specific control temporal control with tamoxifen

  10. NEO Conditional gene activation Inactive allele loxP sites CRE Cell-specific promoter Active allele loxP site

  11. NEO Conditional gene activation Inactive allele loxP sites CRE Cell-specific promoter Active allele loxP site • Reporter gene • Ion channel • GPCR • Toxin

  12. Targeted introduction of a mutation NEO NEO mutation Targeting construct Inactive allele loxP sites CRE mutation Active allele with mutation loxP site

  13. NEO Conditional exon swap (double recombination) Frt site Lox P site Lox P site (different) Targeting construct Remove frtNeo with FLP recombinase 2-step recombination only between like lox P sites following path a or b Double recombination with Cre recombinase a b a b

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