Targeting projects at PolyGene are processed along a scheme in the below order. As a customer, you will receive a report, and be billed, as each milestone is achieved, and on milestones with a longer duration, receive a bi-weekly progress update. Extensive warranties are given as indicated in the quote, some of which however dependent on processing here (in the case our task is to provide partial services).
For the different types of projects (KO, KI, conditional KO), the steps are similar, and thus, are similarly priced.
Strategizing 1 month
- Starting from the accession number we analyze the gene of interest based on literature and database research (genomic locus, gene structure, protein structure, expression profile, known knock out models)
- Offer different targeting strategies, including construction of the targeting vector and Southern blot strategy
- Construction of an adequate probe, screening and isolation of the clone of your choice (e.g. from genomic BAC or cDNA libraries), and verification of your clone inserts by
Southern and/or PCR technology - Generation sequences via total gene synthesis that are not otherwise available
Vector construction 2-4 month
- construction of the targeting vector for transfection of embryonic stem (ES) cells
- (PCR-) control vector construction
- establishing the targeting and PCR screening strategies
Embryonic Stem Cell (ES cell) Transfection 2 month
- preparation of the targeting vector
- establishment of the screening procedures (Southern or PCR)
- electroporation of the targeting construct and selection for antibiotic resistance
- isolation and screening of ES cell clones (129Sv or C57Bl/6 derived)
- expansion and verification of positive clones (Southern or PCR)
- karyotyping and morphological clone description
Positive ES cell clones are guaranteed, providing the targeting vector is a PolyGene construct.
Blastocyst Injection 2-4 weeks
Chimeric mice are generated by injection of the embryonic stem cells into C57Bl/6N blastocysts (for C57Bl/6 ES cells: into C57Bl/6grey):
- isolation of blastocysts from donor mice
- injection into blastocysts
- transfer into IVC-held foster mice
- progeny generation and coat colour determination
Breeding 2 months
Chimeric mice are bread to C57Bl/6 and/or Flp-deleter mice depending on the targeting vector for germ line transmission of the targeted allele and excision of the resistance cassette in vivo. The service includes:
- breeding to IVC-heldfoster C57/Bl/6 or deleter-Flp mice
- progeny generation and coat color determination
- design and establishment of a genotyping PCR
- detection of germ line transmission by genotyping PCR