The NIEHS knock out core (KOC) is a service orientated facility, which aims at helping investigators at NIEHS develop animal models to further their research and fulfilling the mission statement of NIEHS. The KOC aids investigators in generating transgenic mouse models specific for their research needs; including traditional knockouts, conditional knockouts, and knock-ins. It also provides assistance in establishing mutant cell lines for in vitro studies, dissecting early stage embryos, and establishing genotyping strategies for both novel mouse lines and existing lines. Each project in the core is a long-term commitment, and numerous projects are at different stages of completion at any given time of the year. The core typically works closely with investigators from project inception, which starts with meeting with the PI's group to determine not only technical aspects of the proposed project but also to provide scientific guidance in generating the most appropriate mouse model. The KOC hired a molecular geneticist to aid investigators that lack expertise in generating targeting vectors and developing screening strategies. With the increased personnel, the KOC has been able to generate transgenic mouse lines using the ROSA26 locus with substantial success. This has been done through conventional homologous recombination and recombinase mediated cassette exchange (RMCE), which allows for insertion of single copy transgenes into the ROSA26 locus. Although the KOC is focused on the scientific support of investigators at the institute, this is done with financial consideration, which includes exploiting the ROSA26 locus to generate classic transgene mouse lines in-house, being available to meet with investigators to optimize mouse breeding strategies, and overseeing complex mouse colonies such as the COX-2 colony, where closely working with CMB and guiding them to develop appropriate screening and breeding strategy to maintain the desired mouse colony saved substantial amount of resources for the DIR. The KOC recently brought in the latest gene editing tool, CRISPR-Cas9 technique to the institute and successfully developed mutant mice as well as mutant cell lines for investigators to aid to their in-vitro analyses, while working on improving homology directed repair (HDR) to introduce small mutations into the genome using this technique. In parallel, the KOC is using this CRISPR-Cas9 tehchnique to target ES-cells via transfection or electroporation with substantial success to introduce point mutation or knock-ins. CRISPR/Cas9 is a revolutionary technique and has tremendous future potentials not only to introduce mutation into the mouse genome to develop mouse models to study in-vivo gene function but curing a wide range of human diseases once it overcomes some of the major hurdles it currently faces. In particular, the off-targeting events that happen to each targeting experiment using Cas9 and the guide RNA, which might have severe deleterious effects on phenotypic analyses. Therefore, the KOC is diligently working on fine-tuning the technique using different ratios of Cas9 to template RNA, as well as choosing alternate and best possible (least matching to the genome sequence) guide RNAs to introduce the desired mutation and minimizing the off-targeting at the lowest level during microinjection. At the same time, the KOC is working on improving CRISPR/Cas9 targeting in ES-cells, which turned out to be extremely efficient comparing to the traditional homologous recombination and helping investigators in targeting genes to develop their mutant mouse models without compromising the time delays, when comparing to microinjection of RNA into one cell embryos (CRISPR). The KOC took several initiatives in recently in an effort to a substantial savings of NIEHS budget and time and bringing in currently stored embryos at Charles River/Jackson Lab/Taconic to a local off-site storage facility with one set to be stored on site as a back up and worked out the detail logistics with the appropriate parities involved. The KOC recently assumed the responsibility of cryopreservation with saving of time and cost with anticipation of major impact on animal husbandry costs for the DIR in the near future. In addition to meeting investigators need to generate mutant mouse models, KOC works closely with CMB/ACUC and provides the necessary expertise to help developing efficient breeding strategies to optimize animal use and minimize the cost. KOC has recently expanded its in-house embryo storage system to CMB for long-term storage of orphaned mouse lines and short-term storage of recently shipped embryos for cryo-recovery. This has reduced the total DIR cost by discontinuing yearly storage payment for low priority and orphan embryo lines from Taconic, Charles River, and Jackson Lab. Additionally, the KOC recently assumed the role of cryo-recovery and embryo transfer from CMB QAL. This resulted in cost savings to NIEHS and faster importation of mouse lines into NIEHS from non-commercial source. In another attempt to help investigators and saving money for the institute the KOC recently adopted the in-vitro fertilization (IVF) technique and will provide the service as needed to DIR. There are several reasons to add IVF to the KOCs list of services. IVF, when combined with Hyper-ova techniques, gives us the ability to generate a large number of 2-cell embryos for cryopreservation using a very limited number of female mice. This not only saves in animal costs, but can also be extremely important when a line has been allowed to languish to just a few mice. In addition, it has been a practice of CMB for last several years to cryopreserve mouse lines using only sperm. If it becomes necessary for any of these lines to be resuscitated, IVF is the necessary technique. Core wont be freezing sperm (CMB is doing that and helping the investigators) but will help recovering the line as a large number of investigators own many of their lines as frozen sperm and this would help in terms of time and saving money for the institute. The KOC provides services to the NIEHS research community across the DIR and has established exceptionally strong research collaboration with DIR investigators. In addition to the cores central mission, the KOMC is actively involved in the analyses and support of previously generated genetically modified alleles. We readily provide support to investigators to develop efficient breeding strategies to generate genetically appropriate experimental cohorts. We work closely with animal facility staff and outside vendors to minimize waste and delays for NIEHS investigators. We routinely help investigators to acquire and validate ES-cells from outside sources (KOMP, EUCOMM, Taconic, and extramural collaborators). Finally, NIEHS KOC played a significant role in providing research support to the DIR investigators through close collaborative efforts that resulted in helping them publishing their research articles in many of those quality peer reviewed journals.

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