How should I contact the Rodent Genome Engineering Lab (RGEL)?

• For general questions, contact RGEL’s Director Dr. Sang Kim at Sang.Kim@NYULangone.org
• For questions regarding generation of complex genomically engineered mouse models, contact Dr. Ran Brosh at  Ran.Brosh@NYULangone.org
• You can also call us at 347-802-5486

What is the rederivation process for transgenic mice (live mice or frozen stock)?

Mouse rederivation is a laboratory technique used to eliminate pathogens from a mouse strain by re-establishing the strain in a sterile or pathogen-free environment. It is required when importing mice from most other facilities. The procedure involves isolation of early, pre-implantation stage embryos from the reproductive tract of 1-2 donor females and their transfer to the reproductive tract of a sterile recipient females which are made pseudo-pregnant in order to allow implantation, gestation, and birth.  Once RGEL re-derives pups, DCM performs serology tests to ensure sterility before mice can be transferred into investigator’s mouse room.  Timeline is 4-5 weeks.

How many male mice are required for sperm cryopreservation and how should I submit a request?

For sperm cryopreservation we require 2 male mice aged between 4-12 months. The PI should initiate a transfer to RGEL’s mouse room (SB 319) on the Animal Operation System (available via Research Navigator). In addition, the PI should submit an iLab “sperm cryopreservation” service request.

How many male and female mouse are required for embryo cryopreservation and how should I submit a request?

Embryo cryopreservation involves super-ovulating female mice and mating them with male stud mice provided by the investigator. RGEL requires 2 males aged between 4-12 months and 5-7 females aged between 1-6 months. ) mice and required transfer into Core mouse room SB 319. The PI should initiate a transfer to RGEL’s mouse room (SB 319) on the Animal Operation System (available via Research Navigator). In addition, the PI should submit an iLab “embryos cryopreservation” service request.

Cryopreservation of embryos is recommended for homozygous lines or lines with multiple genetic alterations.

For a combined sperm and embryo cryopreservation please submit 2 males and 5 females and request both procedures on iLab.

How do I submit DNA for transgenic mice production via zygote microinjection?

For DNA microinjection into fertilized eggs (zygotes) we require 50 µg of digested unpurified DNA. Please follow this procedure:

1. Set up a large digestion reaction with 50 µg of your plasmid DNA in a final volume of 100-150 µl
2. Incubate the digest according to the restriction enzyme manufacturer’s instructions
3. Run ~5 µl of the digested plasmid on an agarose gel to check for successful and complete digest
4. Take a picture of the gel and mark the band that should be purified for microinjection
5. Upload the annotated image when you fill out the request on iLab
6. Also upload the plasmid’s map (preferably in a SnapGene format)
7. Bring the remainder of the digest (in a final volume of 100 to 150 microliters) to RGEL (Room SB 447, science Building 4th floor).
8. We will purify the DNA for microinjection from the digest using the NucleoSpin kit (Macherey-Nagel)

Additional notes:

9. If you want use large DNA fragments such as bacterial artificial chromosomes (BACs), that there is a different protocol for preparation of the BAC DNA for microinjection.
10. DNAs are purified and microinjected into C57BL/6j mouse eggs and surgically transferred to recipients. Other mouse strains can be used for transgenic production when prior arrangements are made. Please contact Sang.Kim@NYULangone.org for inquiry.

How do I prepare traditional gene-targeting vectors for genome engineering in mouse embryonic stem cells?

When submitting DNA constructs for mESC engineering:

1. Consult with the core regarding how to design gene-targeting vectors and how to linearize them
2. Prep the targeting vector (and any additional vector required) using a DNA midiprep or maxiprep kit (not miniprep).
3. Make sure the final volume is at least 1 µg/ml
4. Digest at least 50 µl of plasmid DNA with a restriction enzyme that linearizes the plasmid (ask for help choosing one if you’re not sure)
5. Submit an iLab request for “Gene Targeting in Mouse ES Cells – Electroporation”
6. Drop off your linearized targeting vector (Room SB 447, science Building 4th floor) along with the submission form

How do I design and generate complex mouse models?

RGEL applies state-of-the-art genome engineering methods in mouse embryonic stem cells (mESCs) for generating a wide variety of mouse models, including gene insertions, conditional alleles, large deletions and more. This process can be complex, involving many procedures that are carried out over a period of several months or more. Careful attention to detail in the design stage often makes the difference between a smooth, successful experience and a failure. Investigators are invited to contact Dr. Ran Brosh, an expert in mESC genome engineering and RGEL’s Associate Director at Ran.Brosh@NYLangone.org for help with designing the best genome engineering strategy.

What kind of support can I get for genotyping my transgenic mice?

Please contact Dr. Ran Brosh, RGEL’s Associate Director, at Ran.Brosh@NYULangone.org for help in planning genotyping assays or interpreting genotyping results. In addition, if you need tail tip DNA from wild type C57BL/6 mice (or other strains) to set up controls for your assays, please let us know.