Mutation verification, allele structure and the conversion to conditional knockouts Lydia Teboul Head of Molecular and Cellular Biology MLC Summary Initial structure of alleles used in IMPC. Quality control of ES cells and mice: karyotype and allele structure. Conversion of KO first into conditional and definitive KO alleles: tm1a, b, c and d. Allele structures and phenotypes. The IMPC allele: KO first tm1a loxP Ex n-1 lacZ loxP Neo loxP Critical Ex (n) Ex n+1 The KO first is a genetrap allele with downstream cassettes flanked by loxPs. Allele product is a fusion transcript with lacZ allowing for expression and lineage tracing studies in tissues. The IMPC allele: KO first • Promoter driven selection cassette tm1a Ex n-1 loxP lacZ loxP Neo loxP Critical Ex (n) Ex n+1 • Promoterless selection cassette tm1a Ex n-1 loxP lacZ Neo loxP Critical Ex (n) Ex n+1 Either promoter driven and promoterless selection cassettes have been used to build the collection. Why are we bothering with QC? The IMPC ES cell collection was generated in a high throughput fashion …therefore materials obtained from repository require further quality controls of - karyotype - integrity of targeted allele A series of quality control assays are used to validate ES cells Karyotyping of ES cells: Switch to ddPCR JM8 ES cells have been extensively passaged in the process of generating the mutant collection. Clones show high frequency of trisomy 1, 8 or 11. Trisomic clones will not yield germ line transmission. Trisomy Chr 8 Chromosome spread ddPCR Karyotyping of ES cells: Switch to ddPCR 30% of the clones we received in 2014 were trisomic for at least one of the chromosomes profiled. Verification of loxP presence loxP Ex n-1 lacZ loxP Neo loxP Critical Ex (n) Ex n+1 Universal primers Gene specific or universal PCR & sequencing to detect the 3’ LoxP Gene specific Confirm identity of the targeting construct? Confirm presence/absence of downstream LoxP site? High throughput Southern blotting n’ kb (lacZ or neo) m’ kb (neo) loxP Ex n-1 lacZ 5’HA loxP Neo loxP Critical Ex (n) Ex n+1 3’HA n kb (lacZ) m kb (neo or lacZ) Confirm desired locus has been targeted? Confirm structure of the targeted allele? Confirm absence of additional vector insertions? QC of alleles in mice: Genotyping by copy counting • Full automation of qPCR onto 2 TECAN robots and 384 qPCR machine. BP-LOA NEO LACZ CRE CR LOA • Copy counting of lacZ/Neo cassette and WT alleles (loss-of-allele assay). • Unambiguous allele specific profile: Any mix-up between stocks or heterogeneity in line would be picked up. Tm1a-het Tm1b-het WT Allele quality: all lines are controlled to the highest standard ES cells (2014, ~800 clones) Mice Genotyping by allele counting Amplification + sequencing target region >99% pass rate for F1 mice Southern blotting for imported stocks !!! 84% pass rate for imported lines Allele conversions: tm1a loxP Ex n-1 lacZ Flp loxP Neo loxP Critical Ex (n) Ex n+1 Cre tm1b Ex n-1 tm1c Ex n-1 Critical Ex (n) Cre tm1d Ex n-1 Ex n+1 Ex n+1 lacZ Ex n+1 Allele structures and phenotypes • tm1a and tm1b phenotypes can differ • Example were tm1a phenotype is less severe than tm1b: Jmjd5tm (tm1a data from O.Wendling, ICS) tm1a/tm1a +/+ tm1b/tm1b tm1b/+ Leaky gene trap? -> Rich allele series Allele structures and phenotypes • tm1a and tm1b phenotypes can differ • Example were tm1a phenotype is more severe than tm1b: Afmidtm 45 40 35 Measure of free fed glycemia 30 25 20 15 10 5 0 WT AFMID TM1A AFMID TM1B Disturbed spacing or selection cassette promoter interferes with locus function? Dosage effect? Summary tm1a Ex n-1 lacZ Neo Critical Ex (n) Ex n+1 IMPC alleles are versatile: they can be employed as null or conditionals. Genetrap component allows for gene expression and lineage tracing studies. The different versions of a targeted locus constitute potentially rich allelic series. The large-scale mouse production programme at Harwell includes a comprehensive quality control of mutated alleles. We have the capacity of converting KO first into conditional alleles or definitive KOs on demand. Thank you for your attention Molecular Biology Team Gemma Codner Adam Caulder Lauren Chessum Joffrey Mianne Jorik Loeffler Adam Radage Ted Evans Colin Beechey Microinjection Team Martin Fray Deb Bogani Wendy Gardiner Phenotyping data James Cleak Michelle Stewart Olivia Wendling, ICS, Strasbourg Questions?
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