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Genetically engineered mouse (GEM) models are commonly used in biomedical research. Generating GEMs involve complex set of experimental procedures requiring sophisticated equipment and highly skilled technical staff. Because of these reasons, most research institutes set up centralized core facilities where custom GEMs are created for research groups. Researchers, on the other hand, when they begin thinking about generating GEMs for their research, several questions arise in their minds. For example, what type of model(s) would be best useful for my research, how do I design them, what are the latest technologies and tools available for developing my model(s), and finally how to breed GEMs in my research. As there are several considerations and options in mouse designs, and as it is an expensive and time-consuming endeavor, careful planning upfront can ensure the highest chance of success. In this article, we provide brief answers to several frequently asked questions that arise when researchers begin thinking about generating mouse model(s) for their work. [ABSTRACT FROM AUTHOR]

Targeted transgenic mouse models, where an exogenous gene is inserted into a specified genomic locus to achieve its stable and reliable expression, have been widely used in biomedical research. However, the available methodologies for targeted insertion of sequences require many laborious steps that involve the use of embryonic stem (ES) cells. We recently developed Pronuclear Injection-based Targeted Transgenesis (PITT), a method that uses a recombinase-mediated cassette exchange (RMCE) to enable insertion of sequences at a predetermined genomic locus, such as ROSA26 . The PITT technique uses fertilized eggs (instead of ES cells) collected from ‘seed mice’ that contain the RMCE landing pad. The PITT method can rapidly generate reliable targeted transgenic mice; it requires a seed mouse, which in our previous study was generated using ES cell targeting approaches. Here, we demonstrate that seed mice containing the RMCE landing pad can be developed rapidly by using the CRISPR/Cas9 system. One of the CRISPR targets tested in this study enabled the insertion of sequences precisely at the original ROSA26 provirus integration site. We anticipate that using a similar approach, PITT landing pad sequences can be rapidly and precisely inserted at other genomic loci to develop an array of PITT tools. This two-step strategy combines the best features of the two newer technologies—rapid creation of PITT landing pads using the CRISPR/Cas9 system and efficient and precise insertion of larger cassettes at the landing pads using PITT. This study also revealed that anomalous and mosaic sequence insertions can occur with the CRISPR/Cas9 system. [ABSTRACT FROM AUTHOR]

Genome editing using the CRISPR/Cas9 system requires the presence of guide RNAs bound to the Cas9 endonuclease as a ribonucleoprotein (RNP) complex in cells, which cleaves the host cell genome at sites specified by the guide RNAs. New genetic material may be introduced during repair of the double-stranded break via homology dependent repair (HDR) if suitable DNA templates are delivered with the CRISPR components. Early methods used plasmid or viral vectors to make these components in the host cell, however newer approaches using recombinant Cas9 protein with synthetic guide RNAs introduced directly as an RNP complex into cells shows faster onset of action with fewer off-target effects. This approach also enables use of chemically modified synthetic guide RNAs that have improved nuclease stability and reduces the risk of triggering an innate immune response in the host cell. This article provides detailed methods for genome editing using the RNP approach with synthetic guide RNAs using lipofection or electroporation in mammalian cells or using microinjection in murine zygotes, with or without addition of a single-stranded HDR template DNA. [ABSTRACT FROM AUTHOR]