CRISPR-KO (CRISPR-KO)

The GEM Core will microinject sgRNA and Cas9 mRNA or protein into pronuclear stage mouse embryos for generating founder mice with a mutation, insertion or deletion in the genomic DNA sequence to be targeted. We recommend investigators to use the Mouse Embryonic Stem Cell Core led by Dr. Jason Heaney to design and prepare the sgRNA and Cas9 samples for the injection.

 The GEM Core will inject at least 110 embryos for each request. Each request may have up to two sgRNAs targeting the same gene or genomic DNA locus. If only 0-3 pups are obtained, the Core will repeat the work with 60% discount for samples from MESCC or with 40 percent discount for samples from others. The targeting efficiency by this method is relatively high. However, since the final outcomes of this kind of experiments are dependent on so many different factors, the GEM Core is unable to guarantee a positive outcome.

When the injection mix is prepared by the PI instead of the ES core, there is a Q/C required before the GEM core can accept/process the request. PI makes a small volume of the injection mix (exactly the same as you will make for the actual injections) and aliquots into 2 tubes, one sits in ice and one at room temperature for at least one hour. Then run an agarose gel with equal volume to compare the two samples to check any trace of RNase. Please upload the photo in iLab to this request. The GEM core will check and approve the request if QC is acceptable.

Price: View pricing

CRISPR–KI (CRISPR-KI)

The GEM Core will microinject sgRNA, a single or double strand donor DNA template for homologous DNA repair, and Cas9 mRNA or protein into pronuclear stage mouse embryos for generating founder mice with a pre-designed alteration in a specific genomic DNA sequence. We recommend investigators to use the Mouse Embryonic Stem Cell Core led by Dr. Jason Heaney  to design and prepare the sgRNA, donor DNA and Cas9 samples for the injection.

The GEM Core will inject at least 140 embryos for each request. Each request may have up to two sgRNAs targeting the same gene or genomic DNA locus. The targeting efficiency by this method is much lower than that by CRISPR-KO. Multiple times of microinjection experiments may be needed for obtaining a knock-in mouse line. Since the final outcomes of this kind of experiments depend on so many different factors, the GEM Core is unable to guarantee a positive outcome. Full charge will be applied regardless of how many pups to be obtained, because the viability of the injected embryos is related to the DNA size and sample concentration and purity.

When the injection mix is prepared by the PI instead of the ES core, there is a Q/C required before the GEM core can accept/process the request. PI makes a small volume (about 20 ul) of the injection mix (exactly the same as you will make for the actual injections) and aliquots into 2 tubes, one sits in ice and one at room temperature for at least one hour. Then run at least 5 ul from each tube on a 4% low melting agarose gel with equal volume to compare the two samples to check any trace of RNase. Please upload the photo in iLab to this request. The GEM core will check and approve the request if QC is acceptable.

Price: View detail.