Rodent Genetic Engineering Laboratory Technologies
The Rodent Genetic Engineering Laboratory at NYU Langone is dedicated to using advanced techniques and technologies to produce and manipulate customized genetically engineered rodents. These are conducted on-site in close collaboration with investigators.
Technologies provided by the lab are available to faculty and staff, as well as to investigators from other institutions and laboratories. If you have questions about a service not listed here, contact Sang Y. Kim, PhD, director, at firstname.lastname@example.org.
CRISPR/Cas9 Genome Editing
Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) technology facilitates precision gene editing in rodents. Our lab is exploring, optimizing, and expanding the capabilities of this method for generating mouse models.
We have successfully used CRISPR/Cas9 targeting to produce gene knockouts, conditional knockouts, single-point mutations, and insertions and gene fusions, including simultaneously mutating multiple genes in a single experiment. Mutations can be created by direct injection into C57BL/6 fertilized mouse oocytes as well as into embryonic stem cells (ESCs) in culture. We have also used gene editing to retarget existing strains from repository strains, ESCs, and proprietary frozen sperm using assisted reproductive technology.
Transgenic Mice Generation
Our lab produces traditional transgenic mice by injecting plasmid or bacterial artificial chromosome DNA constructs into a variety of inbred and outbred mouse strains, including C57BL/6. Investigators provide either appropriately purified DNA or 50 µg of vector DNA along with a map and photo documentation of our purification. We guarantee one to four founder mice per construct. We also guarantee that at least 150 eggs are injected and one to four founders, on average, will be generated per plasmid DNA injection daily. Donor females for breeding are provided by the investigator.
Knockout Mice Generation Using Traditional Embryonic Stem Cell Targeting
We produce knockout (KO) mice, conditional knockout (CKO) mice, knock-in (KI) mice, and other types of gene targeting in C57BL/6 ESCs. Our technologies include design support and targeting vector cloning, creation of gene-targeted ESCs, and tetraploid blastocyst microinjection of the ESCs to create fully ESC-derived mice. We can also provide tetraploid blastocyst microinjection of ESCs obtained from collaborators or repositories, including the European Conditional Mouse Mutagenesis Program, the Knockout Mouse Project, and other consortia.
Embryonic Stem Cell Microinjection
We inject ESC clones into a minimum of 50 blastocysts per donor strain. We guarantee at least 5 to 10 chimeric founders per construct that are competent for germline transmission. You provide donor females and any non-C57BL/6 stud males.
Tetraploid Blastocyst Microinjection
We inject ESC-targeted clones or induced pluripotent stem (iPS) cells into the blastocysts of a donor strain. We inject a minimum of 50 blastocysts and guarantee generation of 1 to 10 or more fully ESC-derived offspring. Ten donor B6D2F1 females must be supplied by the investigator. To submit a service request, complete the blastocyst and tetraploid injection requisition form and email it to Dr. Kim at email@example.com.
iPS Cell Microinjection
We inject iPS cells into blastocysts of a donor strain. A minimum of 50 blastocysts are injected. We guarantee 1 to 10 or more fully iPS cell–derived offspring. The investigator provides 10 donor B6D2F1 females.
Gene Targeting in C57BL/6 Embryonic Stem Cells
We target proprietary ESCs. We can also produce ESCs for targeting from mouse strains already present in an NYU Langone vivarium. We provide electroporation, selection, and expansion of targeted clones, using investigator-supplied DNA constructs and genotyping protocols.
Every mouse line imported from a nonapproved vendor or outside institution must be rederived into one of our specific-pathogen-free mouse facilities through embryo transfer. We provide this service in conjunction with the Division of Comparative Medicine (Kerberos ID required for login). We recommend importation of 2 males, 12 weeks old or younger, per line. Wild-type females can be obtained through an approved vendor for a rederivation procedure, or wild-type or mutant females from an NYU Langone vivarium can be used.
Investigators are encouraged to supply fresh or cryopreserved embryos or sperm as an alternative import method, which increases efficiency and reduces time and costs. Contact Dr. Kim for further information.
We freeze and indefinitely store embryos from any strain in liquid nitrogen to reduce cage usage, per diem costs, and the time required to maintain a mouse line. Stored cryopreserved embryos can be a critical backup in case of a pathogen outbreak, a line lost because of breeding cessation, or other unforeseen problems. We can also ship frozen embryos worldwide to collaborators.
Our method of cryopreserving eight-cell embryos using a slow-freezing method in a controlled-rate freezer guarantees viability. The procedure for recovery of strains from frozen embryos is similar to rederivation.
We freeze and indefinitely store sperm from any strain in liquid nitrogen. Transportation of live mice is unnecessary; frozen sperm can be stored long term and later shipped to collaborators. We require 1 actively breeding proven male 14 to 24 weeks old per line for sperm cryopreservation. We reconstitute strains from frozen sperm using in vitro fertilization (IVF).
In Vitro Fertilization
We rescue or expand mouse lines using fresh or frozen sperm in an IVF procedure. Our success rate depends on background strain and sperm quality. We can import mouse lines as frozen sperm and expand them using IVF. We can also attempt to revive a strain that no longer breeds, sometimes even from a newly deceased male. Although we cannot guarantee success for IVF, we are always happy to make an attempt.
We provide tissues for use in preparing DNA for genotyping procedures.
Embryonic Stem Cell Expansion
We offer ESC culture to researchers who purchase or generate MAP-tested targeted ESC clones for blastocyst injection. We strongly recommend karyotyping all injected embryonic stem cell clones prior to injection. Contact Dr. Kim for further details at firstname.lastname@example.org.