Gene Targeting and Transgenic Shared Resource (GeTT)

Transgenic injection (various backgrounds)

The Gene Targeting and Transgenic (GeTT) Shared Resource at Roswell Park Comprehensive Cancer Center will harvest and inject the pronuclei of oocytes with DNA prepared and purified by the investigator or the GeTT (whichever is chosen). Surviving eggs will be implanted in pseudopregnant females, and pups born will undergo tail biopsy for isolation of DNA and will also be identified by an ear tag. The GeTT guarantees that at least 100 eggs will be surgically transferred, or three transgenic offspring will be produced (whichever comes first).

Note: The expression of your gene is not guaranteed.

Lentiviral transgenics

Lentiviral construct injections are performed upon request. Investigator will need to have prior approval from the Institute Biosafety Committee to begin injections. Please contact Aimee Stablewski at 716-845-5843 or Aimee.Stablewski@RoswellPark.org for details.

Upon receiving your lab’s DNA construct, the Gene Targeting and Transgenic (GeTT) Shared Resource at Roswell Park Comprehensive Cancer Center will perform an electroporation of your DNA into W4 (129S6/SVEvTac), G4 (129/B6) or JM8A3.N1 (C57BL/6Tac) mouse embryonic stem cells. Colonies will be selected with the appropriate drug, depending on your positive selectable marker. Colonies also can be negatively selected for enrichment purposes. Selection methods should be discussed with the resource prior to the electroporation date to prepare the necessary feeder cells.

The resource will pick and freeze ~240 colonies, of which we will give the investigator’s lab ~200 DNA preps. The investigator will screen these clones by Southern analysis and/or PCR and notify the resource of the positives for homologous recombination. The facility will expand five of these clones and give the investigator DNA again to reconfirm positives. When reconfirmed, the resource can begin gene targeting microinjections for the investigator's laboratory.

The entire electroporation process takes approximately one month and the resource expects the investigator’s lab to perform the positive confirmations in a timely fashion.

Please contact Aimee Stablewski at 716-845-5843 or Aimee.Stablewski@RoswellPark.org for a project specific cost estimate.

Blastocyst microinjection with your ES cells generated in-house or by a collaborator

The Gene Targeting and Transgenic (GeTT) Shared Resource at Roswell Park Comprehensive Cancer Center will harvest blastocysts from C57BL/6 mice (currently C57BL/6 albino mice from the Jackson Laboratory) and inject the appropriate number of embryonic stem cells/blastocyst (either provided by the investigator or generated in our facility. (If not generated in our resource, you must submit a Mycoplasma Pathology Report and IMPACT testing from IDEXX RADIL with your cell line or we can submit your cell line for testing, which will incur additional charges.)

Injected blastocysts will then be implanted into pseudopregnant females. The GeTT guarantees that at least 20 blastocysts will be successfully injected and implanted into foster mice or three chimeric pups will be produced, whichever comes first.

KOMP/EUCOMM ES cell injections

Same as Blastocyst Microinjection, however, please read this message regarding the KOMP/EUCOMM ES cell injections.

Please contact Aimee Stablewski at 716-845-5843 or Aimee.Stablewski@RoswellPark.org for a project specific cost estimate.

Frozen or fresh embryos

The Gene Targeting and Transgenic (GeTT) Shared Resource at Roswell Park Comprehensive Cancer Center will rederive strains from fresh or frozen embryos at the request of the investigator.

The resource will also work with investigators to rederive lines from dirty facilities into the clean facilities here at Roswell Park. In this situation, arrangements will need to be made with the resource regarding where the mice are currently housed and how the resource will receive embryos for transfer.

In vitro fertilization

Embryos will be created and then rederived as above. Please see IVF for details.

Note: After surgical transfers of washed embryos are done, per diem charges at the GeTT’s rate will be charged to investigators.

Please contact Aimee Stablewski at 716-845-5843 or Aimee.Stablewski@RoswellPark.org for a project specific cost estimate.

Cryopreservation of sperm

Two male mice between the ages of 10-16 weeks are needed to perform this service. We will freeze down 20 straws worth of sperm and will store them in liquid nitrogen. A post-thaw viability check and an in vitro fertilization (IVF) to the 2-cell stage will be performed on one of the straws to determine fertilization rate.

Sperm cryopreservation presents several opportunities:

• Substantially reduce the number of mice in an investigator’s colony
• Sperm collected from a single male can potentially give rise to large numbers of offspring following IVF of oocytes. A large cohort IVF can give an investigator age-matched and sex-matched progeny that are sometimes needed for experimental cohorts and can help to give the number of mice needed when there are multiple alleles needed in the mouse

The disadvantage of sperm cryopreservation is that monoploid genome is preserved. The sperm cryopreservation service does not guarantee recovery to live born by IVF, and for some strains ICSI is required for recovery. Sperm counts, motility and morphology will be analyzed before cryopreservation.

Cryopreservation of embryos

Our professional staff will determine the quantity of embryos to cryopreserve and the most efficient procedure for embryo collections. In vitro quality control to indicate the success of cryopreservation is performed on every batch of embryos.

Embryo collections can be done one of two ways:

• Traditional mating, collection and freezing: Specifically, stud males are mated weekly with egg donors per cryopreservation session to produce embryos for cryopreservation. On average, it takes two to four embryo collection sessions to freeze down enough embryos to guarantee recovery in this traditional way. If using homozygous males, fewer sessions will be needed. However, it may take more sessions if the mouse strain has a low superovulation rate, the stud males have low fertility, or males are too old. Some strains cannot be successfully cryopreserved. The investigator is responsible for providing all stud males, embryo donors and per diem costs for these animals. Females will be superovulated by the GeTT facility and mated with males, and the resulting embryos will be harvested and cryopreserved. This procedure will be repeated until a sufficient number of embryos are cryopreserved. Embryos are isolated and cryopreserved in straws and stored in liquid nitrogen.
• IVF, collection and freezing: Females will be superovulated by the GeTT and embryos will be created through IVF with frozen or fresh sperm. Embryos will be cryopreserved at the 2-cell stage. This procedure will be repeated until a sufficient number of embryos are cryopreserved. This usually should only take one session, but sometimes a second is needed. Embryos are cryopreserved in straws and stored in liquid nitrogen.

The GeTT stores embryos in two separate tanks at Roswell Park in Buffalo, NY.

Mouse in Vitro Fertilization (IVF)

This procedure can be used to rapidly expand lines from as little as one male that carries the desired genotype or to maintain strains with poor breeding efficiency. The Gene Targeting and Transgenic (GeTT) Shared Resource at Roswell Park Comprehensive Cancer Center will superovulate egg donors per session. We will perform IVF with sperm from your male and egg donors.

After overnight culture, two-cell embryos will be transferred to pseudopregnant females the following day (unless specified for freezing). All weaned pups will be transferred to the investigator. We expect the investigator to genotype the pups and determine which pups have the desired genotype(s). IVF results vary according to genetic background and the quality of individual males used for IVF. Thus, we cannot offer a guarantee that any given IVF procedure will produce large number of pups.

The GeTT has three sizes of IVF: small, medium and large. We are able to produce up to 100 or more animals at a time if needed.

The Gene Targeting and Transgenic (GeTT) Shared at Roswell Park Comprehensive Cancer Center will prepare new mouse embryonic stem cell lines from blastocysts or mice provided by investigators. Standard methods employing serum-containing medium and MEK1 inhibitor are used. The success rate of this procedure is high provided blastocysts can be obtained from the strain in question. ES cells provide an endless supply of cells for in vitro studies because ES cells do not undergo senescence and cease division as do other cell types (e.g., fibroblasts).

Advance notice should be given, preferably when the future blastocyst donors are obtained. The ideal age for in-house blastocyst donors for superovulation response is 24-28 days for C57BL/6 background.

Please contact Aimee Stablewski at 716-845-5843 or Aimee.Stablewski@RoswellPark.org for a project specific cost estimate.

The Gene Targeting and Transgenic (GeTT) Shared Resource at Roswell Park Comprehensive Cancer Center can provide basic chromosome counting.

Mouse embryonic stem cells, from either your lab or purchased, will be expanded in our facility, frozen down and screened for chromosomal abnormalities via karyotyping.

Please contact Aimee Stablewski at 716-845-5843 or Aimee.Stablewski@RoswellPark.org for a project specific cost estimate.

In certain instances, mouse embryonic stem cells are found to be aneuploid (greater than or less than 40 chromosomes). Aneuploid ES cells might generate chimeras; however, they will never be transmitted through the germline. If you have an aneuploid ES cell clone, it may be possible to rescue that clone by plating it at a low density and picking subclones that would be diploid, and thus transmit through the germline.

Please contact Aimee Stablewski at 716-845-5843 or Aimee.Stablewski@RoswellPark.org for a project specific cost estimate.

CRISPR animal modification

CRISPR/Cas9 is a powerful genome-editing tool that is redefining the boundaries of biological research. The Gene Targeting and Transgenic (GeTT) Shared Resource at Roswell Park Comprehensive Cancer Center is pleased to introduce a full CRISPR-based gene-editing platform to modify your mouse genome to introduce global knockouts, small amino acid substitutions or other small tag knockins as well as larger gene fusions, reporter mice and conditional allele knockins. We have had more than 100 projects since 2013.

Design and production of your guide RNA and oligos:

The GeTT will work with you to design the reagents needed for a successful project or design the entire project for you.

CRISPR electroporation used for global knockouts, small DNA insertions (base changes, amino acid substitutions (≤200bp), large chromosomal deletions, inversions and tranlocations):

The GETT will harvest and electroporate fertilized oocytes with CRISPR reagents. The RNA/protein complex (called an RNP complex) with or without donor DNA are concurrently electroporated and handled with the utmost care to prevent degradation. DNA and RNA are prepared and purified by the GeTT. Surviving eggs will be implanted in pseudopregnant females, and pups born will undergo tail biopsy for isolation of DNA and will also be identified by an ear tag.

CRISPR one-cell injection (used for larger DNA insertions (>500bp):

The GETT will harvest and inject fertilized oocytes with CRISPR reagents. The RNA/protein complex (called an RNP complex) with or without donor DNA are concurrently injected into one-cell embryos and handled with the utmost care to prevent degradation.

Targeting DNA with homologous arms can be co-injected to direct integration/homologous recombination to that site. DNA and RNA are prepared and purified by the GeTT. Surviving eggs will be implanted in pseudopregnant females, and pups born will undergo tail biopsy for isolation of DNA and will also be identified by an ear tag.

The GeTT highly suggests getting targeted next-generation sequencing (NGS) done on the founder and F1 animals resulting from CRISPR injections/electroporations, as mosaicism is always a concern.

CRISPR cell line modification

The Gene Targeting and Transgenic Shared Resource (GeTT) provides services to investigators interested in CRISPR-mediated genome editing of cultured cells to generate cellular models as research tools. Our facility possesses the extensive expertise and technologies for such tasks, including lentiCRISPR, which were perfected through many prior experiments. The types of genetic alterations we can generate include null alleles (KO) and insertion of a reporter cassette (KI), as well as correction of mutations, which can be particularly useful for developing isogenic control cells. We have established an efficient and full-service protocol for these types of need. We will provide free consultation at the beginning of a project. An outline of the experimental steps and the associated cost will then be emailed to the PI before initiation of the experiment. Afterwards, the PI will receive genetically modified polyclonal cells or individually cloned cells.