Gene Targeting Services
Interested parties can contact assistant core director to discuss preliminary approach of intended creation of a mouse model. Primary requirement to generate a mouse model at BCH using mouse core services is the PI must have an approved IACUC and IBC protocol for the desired model. After approval is granted the mouse core will co-ordinate with PI or lab member to submit request form and provide required reagents before scheduling an experiment.
Requirements
- Approved IACUC and IBC protocol for the desired mouse model.
- Submit electronic request form (eForm) for services signed by PI and designated lab member.
- Mouse core will co-ordinate with PI or lab member to collect required reagents before scheduling an experiment.
- A fund number or purchase order number to reimburse fees for requested services.
Do you have a question?
Current Mouse ES Cells Services
Gene Targeting
Gene Targeting vector DNA is constructed by PI and provided to the core to introduce into mES cells by electroporation followed by drug selection. Surviving single clones are picked and grown in 96 wells. One half of replica plates are frozen and the other half is provided for genotyping to identify correct homologous recombination event. After confirmation, positive clones are expanded to prepare frozen stocks and karyotyped for normal chromosome numbers. After verification 2-3 independent clones are recommended for injection into blastocysts to generate chimeric mice. Chimeras are transferred to PI after weaning at P21.
We also offer CRISPR/Cas9 mediated gene targeting in mES cell lines.
Chimeric mice
We have proven experience in generating chimeric mice from various mouse strains by blastocyst injection. We offer C57Bl/6 as standard blastocyst donor strain, however, we provide blastocyst injection using Albino/B6 for additional fees. Imported mES cells must be tested negative for Mycoplasma sp.
Service Facts
- Gene targeting via homologous recombination (HR) using mES cells derived from C57Bl/6 (JM8.A3.N1), F1 (v6.5) or 129Sv (J1) strains. Users can choose number of clones to be picked in 96 – well format and screen colonies for successful recombination event.
- Gene targeting in vitro using CRISPR/Cas9 reagents in also offered as an alternative to microinjection into fertilized embryos.
- Expansion of Homologous Recombination identified positive ES clones
- ES cell karyotyping
ES cell derivation
We offer ES cell line derivation from transgenic, gene targeted or any other mouse models. PI provide males and females to harvest blastocysts and establish ES cell lines. Total number of cell lines is dependent on quality and quantity of blastocysts harvested from a particular strain.
Service Facts
- Embryos at 8-cell stage frozen and stored under liquid nitrogen
- Sperm cryo-preservation performed by harvesting sperm using 2-3 fertility tested males and cryopreserved sperm stored under liquid nitrogen
- Live pups recovered by in vitro fertilization using frozen-thawed sperm
- IVF using fresh sperm for age/sex/genotype matched cohorts
- Re-derivation of imported mouse strains by embryo transfer or IVF