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Design and Generation of Targeting Constructs Using Recombineering-based Method

We design and generate Gene Targeting Constructs. The constructs generated in the CMGM include conventional knock-out, knock-in, conditional knock-out, knock-in subtle mutations such as a point mutation and small deletion, and other custom made constructs.

The constructs are derived from C57Bl/6 genomic DNA, unless investigator provides the BAC clones from other strains. We use C57Bl/6 x 129SVEV F1 hybrid ES cell lines for targeting.

Investigators who plan to make a transgenic/targeting construct please contact the CMGM at gttf@uchc.edu.


What We Need From You:

  • Construct purpose: Conventional or conditional knock-out, point mutation or deletion.
  • cDNA sequence or Genbank accession number.


Declaration:

The service fees cover the materials and technician time. It does not cover the construct design, trouble shooting and consultation during generation of targeting animals. Thus the project should be considered as a collaboration.

Conventional Knock-out (KO)

The KO constructs generated in the GTTF contain:

Conventional Knockout Diagram

Conditional Knock-out (CKO)

The CKO constructs generated in the GTTF contain:

Conditional Knockout Diagram 1

Investigators have following breeding options:

Option 1: Remove the neo cassette by crossing the germline chimeras with a FLP mouse line. The F1 mice will carry a floxed allele as following:

Conditional Knockout Diagram 2

Option 2: Cross the germline chimeras with a Cre mouse line to delete the floxed fragment together with the neo cassette.

Conditional Knockout Diagram 3

Option 3: Cross F1 mice with tissue specific Cre or inducible Cre lines.

Knock-in Subtle Mutations

The point mutation or small deletion mutation constructs generated in the GTTF contains:

Knock-in Mutations Diagram 1

The neo cassette will be excised by crossing the germline chimeras with Cre mouse line to generate the F1 mice carrying the following mutation: