The LRTGT has standardized our protocols for all services. However, we are constantly trying to improve our services. Therefore, we constantly update our protocols. The protocols we currently use are listed below. NOTE: THESE PROTOCOLS WILL BE IN PLACE SHORTLY
In general, we use protocols from two well-known manuals on the subject of generating genetically modified mice. These are:
“Manipulating the Mouse Embryo, 3rd Edition”
Cold Springs Harbor Laboratory Press
Nagy, Gertsenstein, Vintersten, Behringer
“Gene Targeting: A Practical Approach”
Oxford University Press
Edited by Alexandra L. Joyner
Oxford; New York: Oxford Press, c2003
“Methods in Molecular Biology: Gene Knockout Protocols, 2nd Edition”
Volume 530
Edited by Ralf Kuhn and Wolfgang Wurst
Human Press, c2009
In addition, we also recommend you use our standard protocols for DNA isolation, DNA vector preparation, Southern blotting and PCR analysis.
Protocols for Pronuclear Injection and Transgenic Analysis:
- Microinjection of Mouse Zygotes
Protocols for ES cell work:
- Electroporation of ES cells
- DNA isolation form ES cell colonies
- Expansion and freezing targeted ES cell clones
- Freezing drug resistant clones and preparation of replica plates for Southern blot analysis
- Preparation of EMFI cell stocks
Protocols for Blastocyst Injection:
- Collecting zygotes and removing cumulus cells
- Preparation of ES Cells for Injection
- Blastocyst injection
Protocols for Genotyping:
- Isolation of DNA from tail biopsies
- Southern blot analysis
- PCR analysis
Protocols for cryopreservation:
- Recovery of blastocysts
- Embryo Cryopreservation by Slow Freezing
Protocols for rederivation:
- Oviduct Transfer
- Uterine Transfer
Vasectomy for Generation of Sterile Males
