Abstract
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.
Highlights
To date, the transgenic mice generation process largely uses a method in which the DNA is injected into fertilized eggs
Viz. Pronuclear Injection-based Targeted Transgenesis (PITT), the fertilized eggs from the seed mouse are used for injecting a donor plasmid containing the transgenic cassette of interest along with Cre mRNA which results in precise insertion of the transgenic cassette at the landing pad
We tested if a PITT seed mouse can be created at the ROSA26 locus by co-injecting ROSA sgRNA, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated 9 (Cas9) mRNA and a donor-oligo into fertilized eggs
Summary
The transgenic mice generation process largely uses a method in which the DNA is injected into fertilized eggs (pronuclei). Integration of the transgene in a region that codes for important genes might disrupt their function Because of such reasons, typically many independent transgenic lines are screened through strenuous and time taking steps of breeding them to identify the best suited line for further experiments. To overcome the pitfalls of random transgenesis, certain labs have taken the embryonic stem (ES) cell approach to target a single copy of a transgene into well-studied genetic loci, such as ROSA26. This approach involves laborious and time-consuming steps, such as targeting vector construction, ES cell targeting, screening and expansion of correctly targeted clones, chimera generation and breeding them for germ line transmission. Despite the lengthy and expensive process that takes 7–8 months or longer, success is not guaranteed [3] and not surprisingly, this method is not routinely used
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
