Targeting Services (Mouse)

PolyGene provides a complete range of gene targeting mouse services, which are offered on a modular basis. Projects are split up into milestones, each of which is payable in two installments (30% of the sub-project value upfront, and the remaining 70% upon completion and acceptance of the results).

To request a quote, please use our quote request form.

Gene Cloning

Starting from the accession number, our molecular biology staff can reliably:

 construct an adequate probe, and screen and isolate the clone of your choice
   (e.g. from genomic BAC or cDNA libraries), and verify your clone inserts by
   Southern and/or PCR technology
 screen databases for publicly available clones (BAC, cDNA), and retrieve them
 generate sequences via total gene synthesis that are not otherwise available

in approximately 1-2 months time.

Vector Construction

State-of-the-art targeting vectors may be suitable as well for conventional as for conditional gene targeting. The modular services comprise:

 subcloning of DNA (e.g. genomic or cDNA) inserts
 construction of the vector for targeting in embryonic stem cells (ES cells),
 use of a variety of optional strategies, including (but not limited to) Cre/lox and
   Flp/FRT systems for conditional gene targeting
 construction of the (PCR-) control vector,
 establishing the targeting and PCR screening strategies,

and can be performed in approximately 4-8 months, depending on complexity.

Embryonic Stem Cell (ES cell) Transfection

Targeting constructs are transfected into a variety of ES cell strains. The service includes:

 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 (typically 388),
 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.

Clones, ready for blastocyst injection, are deliverable within 3 months.

Subsequent Recombination

In conditional targeting, the elimination of an antibiotic selection cassette can be achieved, and is offered by PolyGene, by

 electroporation or lipofection of ES clones with Cre/Flp expression plasmids,
   followed by subsequent isolation of ES cell clones and screening: or
 elimination of a FRT-flanked selection cassette by breeding with a Flp deleter
   mouse; or
 elimination of a loxP-flanked selection cassette by pronuclear microinjection of
   Cre recombinase

Blastocyst Injection

Chimeric mice are generated by injection of the embryonic stem cells into C57Bl/6N blastocysts (for C57Bl/6 ES cells: into C57Bl/6grey or BALB/c). The service consists of:

 isolation of blastocysts from donor mice,
 injection into blastocysts,
 transfer into IVC-heldfoster mice,
 progeny generation and coat colour determination.

Providing ES cell clones were generated by PolyGene, and in dependence of the ES cell strain germ-line transmitting chimeric offspring can be guaranteed.

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