The Advanced Rodent Transgenics 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 ART-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 Ran Brosh, PhD, at Ran.Brosh@NYULangone.org.

CRISPR/Cas9-meditated Genome Editing via Pronuclear Microinjection

Pronuclear microinjection enables the direct delivery of CRISPR/Cas9 reagents and donor DNA into mouse zygotes to achieve targeted genome modifications such as knockouts (KOs), conditional knockouts (cKOs), knock-ins (KIs), or point mutations. This approach allows for efficient and rapid generation of genetically engineered mouse models to support research objectives. Unlike mESC-based methods, pronuclear microinjection bypasses intermediate cell engineering steps, enabling faster production of founder animals with desired genetic edits.

Traditional Transgenic Mice Generation

Traditional mouse transgenesis by injecting linearized DNA constructs (plasmids or BACs) directly into the pronucleus of fertilized zygotes (typically harvested from superovulated females).

Mouse Embryonic Stem Cell Genome Engineering

Mouse embryonic stem cells (mESCs) are highly amenable to genomic engineering due to their robust DNA repair mechanisms (primarily HDR), efficient self-renewal, and rapid proliferation. Unlike pronuclear microinjections, genomic edits in mESCs can be selected using common positive and negative selection markers and verified PCR genotyping, sequencing, karyotyping, and other assays. Together, these features enable the generation of highly sophisticated alleles in mESCs, including large deletions (KOs), insertions (KIs), conditional alleles, and more. Most edits are achieved by delivering CRISPR/Cas9 reagents to introduce targeted DNA breaks along with repair donors that specify the desired modification. Additional methods include using recombinases, Base Editing, Prime Editing, Recombinase-mediated cassette exchange, etc.

Engineered mESCs are injected into donor blastocyst to generate chimeric male founders, which are bred to WT females (typically albinos) to confirm germline transmission, mESCs can also be injected into tetraploid donor bastocysts to generate a mouse that is fully derived from the engineering cells.

Embryonic Stem Cell Microinjection

We inject mESCs or mouse induced pluripotent (miPSCs) into donor blastocysts and transfer these blastocysts to pseudopregnant recipient foster females. Injected cells are typically derived from C57BL/6 inbred male mice or from C57BL/6x129 hybrid male mice. Donor blastocysts are typically derived from superovulated females of either C57BL/ 6 or C57BL/6-albino (Tyr-/-) mice, depending on the submitted cells. At least 50 blastocysts will be injected per service request. Each blastocyst is injected with 10-15 PSCs. Chimeric pups will be transferred to requesting lab after weaning (age 3 weeks). Injection can also be done with tetraploid blastocysts that cannot form an embryo, leading to a mouse that is fully derived from the injected mESCs. 

Rederivation

Rederivation is a process used to eliminate pathogens from mouse lines for transferring fertilized embryos into specific pathogen-free (SPF) surrogate mothers. This technique ensures that production of offspring free from undesirable infectious agents, enabling the safe reintroduction of mouse lines into clean facilities or maintaining high health standards within existing colonies. All mice imported to NYU Langone's barrier facilities require rederivation unless importated from approved vendors (please read the DCM animal report policy). This service is also offered for external institutions. 

Embryo Cryopreservation and Recovery

We freeze and store embryos from any strain in liquid nitrogen to reduce cage usage, per diem costs, and the time reuqired 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 problem. We can also ship frozen embryos worldwide to collaborators.

Our method of cryopreserving eight-cell embryos using a slow-freezing in a controlled-rate freezer guarantees viability. The procedure for recovery of strains from frozen embryos is similar to rederivation.

Sperm Cryopreservation and Recovery

We freeze and store sperm from any strain in liquid nitrogen. 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 the background of the 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 try.

Tail Biopsy

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 mESC clones for blastocyst injection. We strongly recommend karyotyping all injected embryonic stem cell clones prior to injection.