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

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

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

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

5. 5 NEO Targeted gene knock-in (knock-out) in ES cells NEO CRE 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. Targeting construct NEO CRE NEO CRETK DTa Positive selection Negative selection Note insertion between transcription and translation start sites

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

7. 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) C -------------N ITR promoter gene of interest WPRE pA ITR backwards N -------------C ITR promoter gene of interest WPRE pA ITR backwards DIO FLEX Double recombination results in expression of gene of interest N -------------C First recombination (inversion), intermediate stage Second recombination (deletion), locks it into inverted position

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

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

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

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

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

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