The Beth Israel Deaconess Transgenic Core Facility opened in October of 1991. The first transgenic mouse created in the facility was born in January of 1992. In 1994 the Core became the Cohen-Solomon transgenic laboratory.
The Transgenic Core is a state-of-art facility that produces genetically altered mice, cryopreserves embryos and sperm, and will reconstitute mouse lines from other institutions via IVF from frozen sperm or thawing and transferring frozen embryos. The Core has created over 1500 lines of transgenic mice, cryopreserved over 500 lines of mouse sperm/embryos and reconstituted over 50 lines of mice from outside institutions. The Core has generated transgenics in several lines of mice, but predominantly FVB/N, C57BL/6 and Balb/C.
Since 2017, most transgenic mice produced by the Core have used CRISPR technology. The method is simple and requires little or no molecular biology skills; hence, it is accessible to all labs. Experts from the Lowell lab will guide the investigators in the design of the CRISPR reagents and suggest the best approaches for identifying founders and verifying the absence of mutations in the targeted gene.
The method is as described below:
Using “Easi-CRISPR” (Quadros RM, Genome Biology, 2017, PMID: 28511701), we can now rapidly and easily generate Cre knockin mice, as well as many other types of mice, via one-step zygote injection of gene targeting reagents. Importantly, the homologous recombination process is extremely efficient, even for targeted insertion of large fragments like Cre. This approach bypasses the need to do gene targeting in mouse embryonic stem cells, greatly reducing the work and time needed to create gene targeted mice.
The key advances of “Easi-CRISPR” are: a) use of single-stranded DNA (ssDNA) targeting constructs which greatly increases targeting efficiency, and b) the ability of a commercial supplier to now synthesize long stretches of ssDNA allowing for targeted insertion of large DNA fragments (up to 2 kb, which includes Cre, GFP, a loxed exon, etc.).
The steps are essentially as described below:
Step 1: You select a sgRNA which will direct Cas9 to cut genomic DNA at or near the desired site of targeted insertion.
Step 2: You design, in silico, your targeting construct sequence. To make a knockin p2A-Cre driver mouse, this construct would be ~1,300 bases long and from 5’ to 3’ would include ~100 bases of target gene 5’ homology arm sequence, 66 bases of p2A sequence, ~1,032 bases of Cre sequence, and ~100 bases of target gene 3’ homology arm sequence.
Step 3: A company then synthesizes your “in silico” targeting construct as ssDNA at $0.90/base. See link below:
Megamers Single-Stranded DNA Fragments
Step 4: The BIDMC Transgenic Core then injects a mixture of your sgRNA, your ssDNA targeting construct and Cas9 protein into fertilized zygotes and will perform embryo transfers.
Step 5: Post embryo transfer, gestation is approximately 3 weeks, and tails from the live-born F0 mice are cut at 2 weeks of age. You then isolate genomic DNA and perform PCR across the 5’ and 3’ arms to detect the F0 founder mice with targeted knockin.
Step 6: Identified knockin F0 founders are then ready to breed for further use.
The Quadros reference cited above reports very high efficiency for Easi-CRISPR on many different targets. For example, the Lowell lab has generated p2A-Cre knockin mice using Easi-CRISPR. 12 of 24 live-born F0 mice had p2A-Cre inserted homologously into the endogenous gene. One of the 12 targeted mice was homozygous for the p2A-Cre knockin allele. Clearly, as reported by Quadros et al., the method is extremely efficient. And importantly, it is extremely simple to do – requires no molecular biology skills. Finally, because the 5’ and 3’ homology arms are very short (~100bp), the targeted allele is extremely easy to genotype using short-range PCR.
As mentioned above, the approach can be used to insert Cre, but also many other things including Flp, Dre, XFP, an epitope tag, etc. Also, it can be used to place lox sites around an exon of a gene.
If you are interested in using this approach to generate mice, feel free to contact Bradford Lowell (blowell@bidmc.harvard.edu) and/or Daqing Wang (dwang11@bidmc.harvard.edu). Daqing Wang is the resident expert in the Lowell lab on using CRISPR to engineer mice and can provide detailed advice and guidance on any of the steps mentioned above. Joel Lawitts (jlawitts@bidmc.harvard.edu) directs the Transgenic Core Facility at BIDMC and can answer questions related to “Step 4”.
Guarantee: The success of the project is dependent upon the design of the reagents. Even though investigators will have access to expert advice, periodically, either the sgRNA(s) for ssDNA will need to be redesigned. Therefore, we cannot guarantee founders. However, the Core will provide approximately 50 potential founders to the investigator for analysis. If there are no founders, reagents can be redesigned, and will be reinjected, at an additional cost, which is less than a new project charge.
Pricing: Available upon request. Please contact Joel Lawitts for all pricing inquires.
Applications to use the Core: Please contact Joel Lawitts for all applications.