December 18, 2015 | by City of Hope
Shared Resources and Services
To support City of Hope’s goal of advancing basic scientific discoveries toward clinical practice, the Transgenic Knockout/Animal Mouse Program (TKAP) was established by Jeffrey Mann, Ph.D., in 1994 and is currently led (since 2004) by experienced core director Walter Tsark, Ph.D. The goal of the core is to provide readily accessible, on-site expertise in creating genetically modified mouse models. Such models are often logical extensions of in vitro studies because they provide a model system with greater physiological relevance to human disease. As such, they often represent a critical stepping stone along the path toward the clinic. For over 20 years, the TKAP has been an invaluable resource aiding myriad City of Hope investigators; the core typically supports 10 to 15 users per year depending on the timeline needed to develop an appropriate model.
By leveraging the expertise at the Transgenic Knockout/Animal Mouse Program (TKAP) to develop genetically modified in vivo mouse models, City of Hope investigators may evaluate both normal gene functions and the role genetic mutations play in the etiology/treatment of diseases. The TKAP also supports our researchers with numerous services that include 1) assisting with reproduction for mouse models; 2) helping investigators choose a program-appropriate technology; 3) weighing advantages and disadvantages of potential approaches and developing a timeline to produce the model; 4) re-deriving gametes/embryos from external sources into in-house colonies; and 5) providing cryopreservation of embryos and gametes to help reduce colony sizes and costs for investigators.
Tsark’s interests lie in understanding the molecular, genetic and epigenetic mechanisms involved in regulating and stabilizing differentiation of pluripotent cells into specialized cell types. Tsark received his Ph.D. in cell and developmental biology at the University of California, Davis, where he worked with Gary Anderson, Ph.D., and collaborated with Arthur Riggs, Ph.D., at City of Hope to develop transgenic mice used to study the role of DNA methylation in mammalian development and genomic imprinting. He then joined City of Hope in 1995 as a postdoctoral fellow in the Riggs laboratory working on DNA methylation and regulation of gene expression. This was followed by postdoctoral research with former TKAP director Mann, examining the genomic imprinting of genes. Tsark went on to join the TKAP as a senior research associate; he later transitioned to TKAP manager and then director. The TKAP team includes Marisa McDonald, who has been with City of Hope for over four years and recently joined the TKAP.
To produce a broad array of genetically modified mouse models ranging from transgenic mice to targeted gene modifications (knockout/knock-in), the TKAP has used traditional gene-targeting methods involving pluripotent (i.e., able to differentiate into all tissue types) mouse embryonic stem cells (mESC). The traditional gene-targeting approach involves transferring purified DNA into the mESC, positive-selection to enrich for recombinant ES cell clones, confirming the genotype of each clone and expanding recombinant mESC. The final step in this long process is to use the recombinant mESC to produce chimeric mice that are capable of transmitting the mutation to their offspring. More recently, the TKAP has implemented a less time intensive approach using clustered regularly interspaced short palindromic repeats (CRISPR) guide RNA and the CRISPR-associated protein 9 (Cas9) RNA-guided DNA endonuclease. Base-pairing between the CRISPR RNA and a complementary DNA sequence in the target gene determines the precise location where the Cas9 endonuclease will create a DNA double strand break. Reducing the frequency of “off-target” cleavage in DNA sharing homology with the actual target gene is often accomplished by using a Cas9 variant that nicks one strand of the DNA (Cas9-N) with a pair of CRISPR guide RNAs binding within ~ 20 base pairs of each other on opposite DNA strands. The requirement for nicking at two independent sequences results in significant improvements in “on-target” specificity, and the cellular DNA repair machinery processes the resulting “offset nicks” as a double-strand DNA break. The significant advantage of the CRISPR/Cas9 approach over traditional gene-targeting is that it allows one to easily change the gene target by simply changing the RNA guide, and it can be used to develop a variety of in vitro and in vivo mouse models. Via this strategy, the TKAP routinely uses pluripotent embryonic stem cells and pre-implantation embryos from standard inbred strains of mice (C57 BL/6 and 129S1) to create transgenic and knockout mouse models. The TKAP has also enjoyed success with using the CRISPR/Cas9 technology to create precise mutations in strains of mice for which pluripotent embryonic stem cell lines are not available. Tsark looks forward to providing expertise using these approaches and the experience/services of the TKAP to advance basic science and translational medicine at City of Hope.
For further information about TKAP, contact Walter Tsark, Ph.D.
Review articles on creating mouse models for cancer research using the CRISPR/Cas9 system:
Doudna JA, Charpentier, E. 2014. Science 346(6213):1258096.
Seruggia D, Montoliu L. 2014. Transgenic Res 23(5):707-16.
Mou H, Kennedy Z, Anderson DG, Yin H, Xue, W. 2015. Genome Med 7(1):53.
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