Cre-lox Basics - Generating Knockout Mice Webinar …

1 Cre-lox Basics - Generating Knockout Mice Technical Information Services Upcoming JAX Webinars 2 Humanized NSG Mice for Innovative Preclinical Research o March 24, 2016, 1:00 pm ET USA o March 29, 2016, 9:00 am ET USA Efficient Mouse Colony Management o April 7, 2016, 1:00 pm ET USA Comparing Mouse Models for Obesity & Diabetes Research o April 14, 2016, 1:00 pm ET USA | Subscribe to the monthly Webinar announcements email list: Make Your Life in the Lab Easier JAX Blog 3 | The Jackson Laboratory s Mission 4 Performing Research Investigating genetics and biology of human disease Providing Resources JAX Mice Clinical & Research Services, bioinformatics data, technical publications and Educating Scientists World-class courses, internships and other programs | JAX Mice The Gold Standard for Biomedical Research JAX Mice The Gold Standard for Biomedical Research 5 NIH funded resource >8.

2 000 - strains and growing million mice shipped annually Unsurpassed genetic quality & animal health Best characterized & referenced ~100 new pubs/week Common inbred strains (C57BL/6J, BALB/cJ, DBA/2J) support development/collection of specialty strains and other valuable community research resources JAX Mice | Online Resources to Expedite Research 6 JAX Mice Database Mouse Genome Informatics Mouse Phenome Database And many more unique resources Contact Technical Support | Online Resources to Expedite Research Presentation Overview Basic Cre-lox mechanism Strain types and breeding schemes oTissue-specific knockouts oGeneral knockouts oInducible knockouts oReporters Cre-lox web resources (finding mice, Cre activity data, and more)

3 7 A Revolutionary Genetic Tool Cre-lox system Natural part of P1 bacteriophage viral life cycle Viral DNA injected into bacteria, circularized using Cre-lox , and replicated for development of new viruses Cre 8 Cre-lox Successfully Engineered in Other Organisms Organisms Yeast Plants Mammalian cell cultures Mice 9 Cre-lox Successfully Engineered in Other Organisms Organisms Yeast Plants Mammalian cell cultures Mice Allows Alteration & deletion of DNA Regulation of location and timing of gene recombination 10 A Simple, Two Component System Cre recombinase Site-specific enzyme, catalyzes recombination between two LoxP sites loxP site 34 base pair DNA sequence Location and orientation determines recombination result: oDeletion oInversion oTranslocation Reviewed in: Nagy A.

4 2000. Genesis 26(2):99-109. PMID:10686599 ATAACTTCGTATA-NNNTANNN-TATACGAAGTTAT Abundant possibilities for genome manipulation! Cre 11 Mechanism: Cre-lox Deletion Floxed target gene Knockout allele X GeneX LoxP GeneX LoxP LoxP GeneX LoxP Cre excision 12 Mechanism: Cre-lox Inversion Cre LoxP LoxP GeneX LoxP LoxP GeneX 13 Mechanism: Cre-lox Translocation Cre Chr 3 Chr 6 LoxP LoxP Reciprocal Translocation (3;6) LoxP LoxP 14 Cre-lox Tissue-Specific Knockout LoxP GeneX LoxP GeneX LoxP LoxP Homozygous floxed mouse 15 Alb cre GeneX GeneX x Liver-specific cre transgene Ex: (Alb-Cre)21 Mgn/J (003574) Cre-lox Tissue-Specific Knockout LoxP GeneX LoxP Alb cre GeneX Cre-lox mouse: Heterozygous for gene X conditional Knockout after 1 generation Liver-specific cre transgene Ex.

5 (Alb-Cre)21 Mgn/J (003574) Alb cre GeneX GeneX LoxP GeneX LoxP GeneX LoxP LoxP x Homozygous floxed mouse 16 Cre-lox Tissue-Specific Knockout 17 LoxP GeneX LoxP Alb GeneX cre Cre-lox Tissue-Specific Knockout (continued) LoxP GeneX LoxP GeneX LoxP LoxP Hemizygous Alb-cre heterozygous floxed gene 25% homozygous for gene X conditional Knockout (2nd generation) GeneX Alb cre GeneX LoxP LoxP LoxP LoxP x Homozygous floxed mouse 18 LoxP GeneX LoxP Alb cre GeneX Cre-lox Tissue-Specific Knockout (continued) 19 Alb-cre + loxP + loxP + Alb cre Gene X GeneX Hemizygous cre Gene X GeneX loxP Cre-lox Tissue-Specific Knockout (continued) 20 Alb-cre + loxP + loxP + loxP Alb-cre loxP/loxP Alb-cre loxP/+ + loxP/loxP + loxP/+ Alb cre Gene X GeneX Gene X GeneX Alb cre Gene X GeneX 25% homozygous for gene X conditional Knockout (2nd generation)

6 Hemizygous cre Hemizygous Alb-cre heterozygous floxed gene LoxP GeneX LoxP GeneX LoxP LoxP x Homozygous floxed mouse 21 Alb cre GeneX LoxP GeneX LoxP Improving Conditional Knockout Efficiency Improving Conditional Knockout Efficiency x Heterozygous null mouse (traditional Knockout ) GeneX Alb cre GeneX LoxP GeneX LoxP conditional gene X knockouts LoxP GeneX LoxP Alb cre 22 Hemizygous Alb-cre heterozygous floxed gene Cre-lox Tissue-Specific Knockout (continued) 23 Alb-cre + loxP + loxP + + - Alb cre Gene X GeneX Hemizygous cre GeneX Cre-lox Tissue-Specific Knockout (continued)

7 24 Alb-cre + loxP + loxP + + Alb-cre loxP / + Alb-cre + / + + loxP /+ + + / + - Alb-cre loxP / - Alb-cre + / - + loxP / - + + / - Alb cre Gene X GeneX Hemizygous cre GeneX Alb cre GeneX conditional gene X knockouts Improving Conditional Knockout Efficiency Hemizygous Alb-cre heterozygous null mouse x GeneX LoxP LoxP Alb cre GeneX LoxP GeneX LoxP LoxP GeneX LoxP Alb cre 25% conditional gene X knockouts Homozygous floxed mouse 25 x Homozygous floxed mouse oocyte-specific cre expression Ex: C57BL/6-Tg(Zp3-cre)93 Knw/J (003651) ZP3 cre Cre-lox Germline Knockouts GeneX LoxP GeneX LoxP Ovary ZP3 cre Oocytes 2 more generations to produce homozygote null mouse 26 Homozygous LoxP mouse Cre mouse cre transgene (Tg) early, widespread expression promoter (Sox2-cre)1 Amc/J (008454) LoxP GeneX LoxP LoxP GeneX LoxP Cre GeneX GeneX Sox2 Cre-lox Knockout Breeding Scheme Offspring: 50% heterozygous Knockout after 1 generation GeneX x 27 Offspring 2nd generation.

8 25% homozygous Knockout GeneX GeneX x 28 Cre-lox Knockout Breeding Scheme (continued) Cre-lox Summary Tissue-specific deletion 2 generations of breeding Cre required to maintain line for future generations Genotype of whole mouse: homozygous flox; Cre Tissue-specific genotype: homozygous flox-deleted; Cre Germline/Embryonic deletion 2-3 generations of breeding Cre not required after germline deletion (can breed it out) Genotype of whole mouse, germplasm, organs & tissues: homozygous flox-deleted ( Knockout ) for gene of interest GeneX LoxP GeneX Cre Alb 29 LoxP LoxP LoxP Inducible Cre Mouse Models x Homozygous LoxP mouse Inducible Cre mouse tamoxifen dependent Cre function Ex.

9 (CAG-cre/Esr1*)5 Amc/J (004682) Induce homozygous Knockout of gene X with tamoxifen + Tamoxifen 2 Generations LoxP GeneX LoxP LoxP GeneX LoxP 30 The FLP-FRT System Analogous to the Cre-lox system Derived from the yeast Saccharomyces cerevisiae Becoming more popular in mouse-based research Flipase recombinase (FLP) recognizes a pair of target sequences (FRT) that flank a genomic region of interest 31 Using FLP-FRT with Cre-lox LoxP LoxP PGK-neo exon exon FRT FRT FLPase LoxP LoxP exon exon FRT exon exon 32 Cre Considerations Mosaicism Some target cells may not express Cre, or LoxP sites may not recombine May be integration site specific; evaluate multiple cre transgenic founders Expression in unexpected tissues (including germline) Use Cre-positive males for breeding Test expression using reporter LoxP site recombination efficiency Affected by position and distance Heffner et al.

10 2012. Nat Commun 3:1218. PMID: 23169059 Schmidt-Supprian and Rajewsky. 2007. Nat Immunol 8(7):665-8. PMID:17579640 Cre mosaicism in mouse mammary gland epithelial cells 33 Cre Considerations (continued) Breeding efficiency If possible, have cre transgene on a different chromosome than the floxed allele Often good idea to breed out cre transgene after germline deletion Cre may produce a phenotype by itself Insertion site effects; changes in gene expression Cre toxicity Target gene may be expressed prior to Cre recombination Consider that genetic background may affect phenotype Heffner et al.