Useful Tips

For ES Cell Service:

Recommended ES lines with a very good rate of chimerism and Germline transmission: V6.4 (B6X129 hybrid) or EAP6 (B6N).

For Cryopreservation:

Although more expensive than the sperm cryo service, the embryo cryo is recommended when mouse lines are to be terminated and the P.I anticipates that there will be a significant demand for supplying these lines to other labs. The large number of embryos collected will afford the P.I to share samples with collaborators and still have enough stock to revive the line in the future. The rate of recovery of live embryos at thawing hovers at 80-90%.

The sperm cryo service is preferred for a back-up strategy while the line is ongoing, although it can also be used to preserve lines that are being terminated. We store an average of 75 embryos as part of the sperm cryo service. These are generally reserved for the core use in case the IVF with frozen sperm is not successful to bring back a line. Our success in bringing back lines from frozen sperm samples averages at 62%. Several factors contribute to variable success rates of IVF; among them are: mouse strain, response to superovulation, source of sperm (fresh vs frozen and Core’s storage vs imported).

Progress Update on CRISPR efficiency:

Throughout 2017, the Transgenic & Chimeric Mouse Facility, in collaboration with the CRISPR/Cas9 Mouse Targeting Core, has processed 65 service requests for genome-editing using CRISPR/cas9 microinjection procedure. Of those 61 (94%) projects generated positive founder mice.

For efficiency purposes, we divide those projects into 4 major groups: KO (indels, deletions), KI minor (point mutation, base substitution, tags..), cKO (conditional with LoxP’s), and KI major (large inserts, ~1.0 Kb).

Overall, a KO was detected in 30% of the mice born, KI minor was at detected 27%, and cKO at 32% when accounting for one LoxP insertion and at 4% when detecting the correct insertion of 5’ and 3’ LoxP’s in cis. KI major remains challenging with efficiency at the level of 3%.

We are now working on methodologies to increase the efficiency of the LoxP KI as well as the large fragment KI.

For CRISPR Injection:

Please see added protocols under the TCMF “PROTOCOLS” section.

The Core has been involved in injecting CRISPR’s for several months now. Some projects used purified RNA’s (for guides and Cas9) for cytoplasmic injections while others used DNA plasmids (for in vivo transcription) for pronuclear injections. The success rate of generating positive mice ranged from 10-50% and included KO (few bp to few Kb), KI (short tag) or KO with repair. Some projects were not successful after multiple attempts and they are being revised as we speak.

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