What we do
The mission of the Gene Targeting and Transgenic (GeTT) Shared Resource at Roswell Park Comprehensive Cancer Center is to ensure investigators can mimic disease in vivo by creating genetically modified mouse models. These models allow investigators to conduct research that can lead to new developments and treatments against cancer.
To obtain these mouse models, our team uses highly specialized instrumentation and expertise not available in most labs.
The Resource Director and Co-director provide guidance to investigators from the earliest planning stages of the project when constructs are designed to advanced stages of the project during phenotype analysis. Resource technicians perform the specialized mouse embryonic stem cell and embryo manipulation methods to generate the genetically modified models.
During the past 22 years, the facility has provided more than 1,000 new genetically modified lines. Our staff have generated these models on multiple strain backgrounds, using traditional methods such as transgenics and embryonic stem cell technology and also by newer methods such as CRISPR/Cas9 technology for the four CCSG programs. These mouse models are used to explain pathways contributing to tumor development and progression. This will continue using a variety of genome sequences and the initiation of large-scale efforts to functionally annotate these genomes.
Use of the resource continues to increase with the ongoing recruitment into the Tumor Immunology and Immunotherapy, Cancer Stress Biology, and Developmental Therapeutics programs. Projects requiring the development of genetically modified mouse models increasingly are becoming relevant to validate in vitro findings, especially considering high-throughput clinical-based findings.
As such, the GeTT observed an increasing trend in the use of the resource by clinical researchers as well as basic scientists.
Services & fees
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 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.
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.
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.
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.
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.
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.
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.
- Tissue culture: equipment and materials
- Making Embryonic Fibroblasts (EFs)
- Electroporation of DNA into ES cells
- Routine culture of ES cells
- Picking and expanding ES cell colonies
- Purification of Plasmid DNA for Microinjection
- Lysozyme (CsCl DNA preparation)
- Preparation of BAC DNA for microinjection
- National for Biotechnology Information (NCBI) – A national resource for publications and sequence information
- Mouse Genome Informatics (MGI)
- International Mouse Phenotyping Consortium (IMPC) – Incorporates the IKMC (International Knockout Mouse Consortium project (KOMP, EUCOMM).
- Mouse Genome Sequence – Ensembl is a joint project between EMBL - EBI and the Wellcome Trust Sanger Institute to develop a software system which produces and maintains automatic annotation on selected eukaryotic genomes.
- Mutant Mouse Regional Resource Center – The MMRRC distributes and cryopreserves scientifically valuable, genetically engineered mouse strains and mouse ES cell lines with potential value for the genetics and biomedical research community. They are a national network of breeding and distribution facilities plus an information coordinating center serving together as NIH's premier repository of spontaneous and induced mutant mouse and cell lines. The MMRRC is supported by the National Institutes of Health.
- RepeatMasker – Search for repeats in homologous arms.
- International Society for Transgenic Technologies (ISTT) – Is a non-profitmaking Society, with the following aims: To foster and encourage knowledge generation, discussion, training and education, and the diffusion of the technologies and specific research used for the genetic modification of animals, in particular those aimed at generating and/or analyzing transgenic and mutant animals as particularly useful experimental models in the biology, biomedicine and biotechnology disciplines.
- International Mouse Strain Resource (IMSR) – The IMSR is a searchable online database of mouse strains, stocks, and mutant ES cell lines available worldwide, including inbred, mutant, and genetically engineered strains. The goal of the IMSR is to assist the international scientific community in locating and obtaining mouse resources for research. Note that the data content found in the IMSR is as supplied by strain repository holders.
Location & hoursRoswell Park Comprehensive Cancer Center
Gene Targeting and Transgenic Shared Resource
Medical Research Complex
Elm and Carlton Streets
Buffalo, New York 14263
Monday – Friday, 6:30 a.m.– 5:30 p.m.
This shared resource is funded by NCI P30CA16056. Publications should cite the core grant in the acknowledgment section if publications use data generated by the shared resource. Two copies of the publication acknowledging the core grant should also be submitted to the facility at Elm and Carlton Streets, Buffalo, NY 14263.
The Gene Targeting and Transgenic (GeTT) Resource at Roswell Park Comprehensive Cancer Center enables investigators to create genetically modified mouse models using several approaches for the systematic dissection of the genetic, molecular, cellular and physiological mechanisms underlying complex biological processes. The resource also provides reproductive technology services to investigators to bank and share their valuable strains, to clean their strains of unwanted pathogens, and to rapidly expand their colonies.
In addition, the GeTT recently expanded to provide Colony Management Services to CCSG investigators. This includes breeding and genotyping of colonies to deliver the multigenic mouse cohorts needed for research.
GeTT provides fee-for-service, first-come-first-serve pronuclear injection, gene targeting, blastocyst injection, cryopreservation, in vitro fertilization and rederivation to Roswell Park, The State University of New York at Buffalo (UB), and other academic investigators at a reasonable price with fast turnaround. Our resource is the sole local provider of these services for the Buffalo and Western New York biomedical research community.
We have an extensive record of high efficiency and success in these services.
The importance of this resource can be seen by the increased demand from within the CCSG programs to develop genetically modified samples. Since it was established, 38 program members have utilized the resource and have generated more than 1,000 different genetically modified mouse models.
It is anticipated that utilization of the resource will increase substantially in the next project period due to increased demand from program members, new recruits and the implementation of new services including use of the powerful genome-editing tool platform. Increased demand also is expected due to the need for validation of high-throughput clinical findings from the success of Roswell Park’s Center for Personalized Medicine and clinical-based research. Cancer research based on human genomics stimulates the need for creation of genetically modified samples and underscores the importance of increasing efficiency in developing models of human cancers.
- Zhang B, Li H, Hu Z, Jiang H, Stablewski AB, Marzullo BJ, Yergeau DA, Feng J. Generation of mouse-human chimeric embryos. Nat Protoc. 2021 Aug;16(8):3954-3980. doi: 10.1038/s41596-021-00565-7. Epub 2021 Jul 2. PMID: 34215863.
- Shahini A, Rajabian N, Choudhury D, Shahini S, Vydiam K, Nguyen T, Kulczyk J, Santarelli T, Ikhapoh I, Zhang Y, Wang J, Liu S, Stablewski A, Thiyagarajan R, Seldeen K, Troen BR, Peirick J, Lei P, Andreadis ST. Ameliorating the hallmarks of cellular senescence in skeletal muscle myogenic progenitors in vitro and in vivo. Sci Adv. 2021 Sep 3;7(36):eabe5671. doi: 10.1126/sciadv.abe5671. Epub 2021 Sep 3. PMID: 34516892; PMCID: PMC8442867.
- Timilsina U, Umthong S, Lynch B, Stablewski A, Stavrou S. SERINC5 Potently Restricts Retrovirus Infection In Vivo. mBio. 2020 Jul 14;11(4):e00588-20. doi: 10.1128/mBio.00588-20. PMID: 32665269; PMCID: PMC7360926.
- Hu Z, Li H, Jiang H, Ren Y, Yu X, Qiu J, Stablewski AB, Zhang B, Buck MJ, Feng J. Transient inhibition of mTOR in human pluripotent stem cells enables robust formation of mouse-human chimeric embryos. Sci Adv. 2020 May 13;6(20):eaaz0298. doi: 10.1126/sciadv.aaz0298. PMID: 32426495; PMCID: PMC7220352.
- Elahi S, Holling GA, Stablewski AB, Olejniczak SH. Improved hematopoietic differentiation of mouse embryonic stem cells through manipulation of the RNA binding protein ARS2. Stem Cell Res. 2020 Mar;43:101710. doi: 10.1016/j.scr.2020.101710. Epub 2020 Jan 18. PMID: 31986485; PMCID: PMC7406152.
- Kelkar A, Zhu Y, Groth T, Stolfa G, Stablewski AB, Singhi N, Nemeth M, Neelamegham S. Doxycycline-Dependent Self-Inactivation of CRISPR-Cas9 to Temporally Regulate On- and Off-Target Editing. Mol Ther. 2020 Jan 8;28(1):29-41. doi: 10.1016/j.ymthe.2019.09.006. Epub 2019 Sep 12. PMID: 31601489; PMCID: PMC6952177.
- Hillman JC, Pugacheva EM, Barger CJ, Sribenja S, Rosario S, Albahrani M, Truskinovsky AM, Stablewski A, Liu S, Loukinov DI, Zentner GE, Lobanenkov VV, Karpf AR, Higgins MJ. BORIS Expression in Ovarian Cancer Precursor Cells Alters the CTCF Cistrome and Enhances Invasiveness through GALNT14. Mol Cancer Res. 2019 Oct;17(10):2051-2062. doi: 10.1158/1541-7786.MCR-19-0310. Epub 2019 Jul 10. PMID: 31292201; PMCID: PMC6943826.
- Barger CJ, Zhang W, Hillman J, Stablewski AB, Higgins MJ, Vanderhyden BC, Odunsi K, Karpf AR. Genetic determinants of FOXM1 overexpression in epithelial ovarian cancer and functional contribution to cell cycle progression. Oncotarget. 2015 Sep 29;6(29):27613-27. doi: 10.18632/oncotarget.4546. PMID: 26243836; PMCID: PMC4695012.
- Bhattacharya A, Klaene JJ, Li Y, Paonessa JD, Stablewski AB, Vouros P, Zhang Y. The inverse relationship between bladder and liver in 4-aminobiphenyl-induced DNA damage. Oncotarget. 2015 Jan 20;6(2):836-45. doi: 10.18632/oncotarget.2722. PMID: 25596734; PMCID: PMC4359259.
- Li F, Cheng Q, Ling X, Stablewski A, Tang L, Foster BA, Johnson CS, Rustum YM, Porter CW. Generation of a novel transgenic mouse model for bioluminescent monitoring of survivin gene activity in vivo at various pathophysiological processes: survivin expression overlaps with stem cell markers. Am J Pathol. 2010 Apr;176(4):1629-38. doi: 10.2353/ajpath.2010.090414. Epub 2010 Feb 4. PMID: 20133811; PMCID: PMC2843455.
- Yoon B, Herman H, Hu B, Park YJ, Lindroth A, Bell A, West AG, Chang Y, Stablewski A, Piel JC, Loukinov DI, Lobanenkov VV, Soloway PD. Rasgrf1 imprinting is regulated by a CTCF-dependent methylation-sensitive enhancer blocker. Mol Cell Biol. 2005 Dec;25(24):11184-90. doi: 10.1128/MCB.25.24.11184-11190.2005. PMID: 16314537; PMCID: PMC1316951.