- Construction and design of ESC targeting vectors by BAC Recombineering, with primers and control vector for screening recombinants.
- Cloning of CRISPR guide sequences, production of CRISPR sg RNA via in-vitro transcription, CRISPR founder screening and Allelic cloning.
- Validation of targeted clones by PCR and Southern blotting.
- ESC targeting, selection, PCR screening followed by expanding and freezing multiple clones
- Injection of validated ESC cells to create chimeric mice
- DNA/RNA (CRISPR) Microinjection to produce transgenic mice
- Breeding chimeras and expansion of mouse lines
- Cryopreservation of mouse embryos and sperm
Molecular Biology Services
If a PI lab does not want to produce their own ESC targeting vectors the Molecular Biology team can design and produce by Bacterial Artificial Chromosomes (BAC) recombineering (recombination-mediated genetic engineering) technology a DNA vector for either direct or conditional gene manipulation strategies. BACs of either 129 or C57BL/6 strains can be requested. The design is done in full consultation with the DCI member's lab with a current wait time of about 2 months to start of the project, on a "first come, first served" basis for DCI members. The Molecular Biology team can also provide complete services for CRISPR in vivo editing in mouse embryos. These services include cloning and validation of CRISPR guide sequences, production of CRISPR sgRNA via in vitro transcription, founder screening and subcloning of the individual alleles from founder mice.
Transgenic and Gene Targeted Mouse Service (Transgenic)
The Transgenic team can target three different mESC lines: a 129S6/SvEvTac, hybrid 129S6/C57BL/6N (G4) (preferred), and C57BL/6N. After selection, clones are screened using PCR and up to 18 positive clones are selected, expanded, and frozen. After targeted clones are adequately confirmed by the BAC/REC team or by the PIs lab, the Transgenic team produces chimeras by microinjection. At least two different clones are injected, and multiple high-quality chimeras are usually obtained. To date, all targeting projects have gone germline. For some experiments, the Transgenic team can subsequently modify validated ES clones by a transfection with Cre or Flp plasmids to effect recombination in vitro. The Transgenic service group also produces genetically modified animals by the traditional DNA microinjection method into the pronucleus of one cell mouse embryo. We have also successfully produced several models by CRISPR construct microinjection.
Rodent Husbandry Services
Once DCI members have obtained targeted mice, they have the option of sending them directly to Traci Reddick in Rodent Husbandry Services for breeding and expansion of the colony. Her team can also cryopreserve embryos and/or sperm for long-term storage or distribution to collaborators. Additionally, if a DCI member needs to use existing mutant animals from an off-campus source the rodent Husbandry service team can receive frozen embryos or sperm imported from outside groups, or rederive imported animal lines that do not meet Duke Health standards.
Even after mice have been transferred to PI labs the members of the Transgenic Shared Resource are available for consultation and clarification on advanced PCR screening and the subsequent breeding to other strains to obtain specific recombination of conditional allels.