The generation of transgenic mice via injection of linearized DNA expression cassettes into fertilized zygotes has first been described in the 80s.1-3 This classical approach, however, is known to drive random integration of the vector.4,5 To overcome this shortcoming and optimize transgene expression, embryonic stem cell (ESC)-based strategies emerged. In this approach, the foreign DNA construct is directed to a specific locus in the host genome – with the help of homologous recombination (HR) strategies – to allow ubiquitous expression of an inserted transgene.6 Following transgenesis, those modified ES cell are then injected into blastocysts and subsequently transferred to surrogate mothers to generate chimeric founder animals.7 Though direct editing in zygotes is a possibility, even with site-directed nucleases such as Cas9, we learn from discussions with our customers that researchers tend to stick with editing of ESC’s, especially for the insertion of larger constructs. Nevertheless, several studies have documented the liability of editing ESC’s to set off genetic mosaicism.4,8-10 Such mosaicism can obscure genotyping of founder animals, thereby delaying development time of a genetically engineered model.
Widespread mosaicism in founder mice
Genetic mosaicism is defined as the coexistence of two or more cell lineages with different genotype originating from a single zygote in a single individual.8,9 Interestingly, mosaicism can stem from various mechanisms, including genomic manipulation (i.e. genome editing) and naturally-occurring ones (e.g. chromosome non-disjunction, anaphase lag, endoreplication, mutations emerging during development).8 The greatest challenge - with mosaicism - is the high likelihood for misinterpretation of founder genotypes. In other words, mosaicism renders it difficult to ascertain whether the genotyped cells (be it from tail, bone marrow or spleen) truly harbors the same genetics as the germline itself. As such, genetic mosaicism can lead to the generation of undesirable compound heterozygous animals.11
Frequent "false positive" founders, as a result of genotypic mosaicism
Mosaicisms are often undesirable because they can lead to false-positive genotyping results. For instance, a founder mouse might display a homozygous deletion based on tail DNA genotyping, but in fact never transmitted the deletion allele to their offspring. With that said, the reverse can also be true (i.e. false-negative genotyping results), whereby pups are falsely identified as negative - via tail lysate genotyping – when they could actually be positive for the engineered allele in the germline. Therefore, genetic mosaicism can surely muddle with phenotype analysis in F0 animals.
Achieving efficient germline transmissions and obtaining true heterozygous and homozygous mutants for functional analyses
On this account, it is important to treat the founders (derived from pronucleus injection-based editing) as chimeras and to make them undergo additional breeding in order to obtain true heterozygous and homozygous mutants for functional analyses.12 As for transgenesis via ESC-based methods, it is recommended to screen many mosaic F0 embryos to achieve germline transmissions harboring the sought-after mutations.13 In such cases, evaluating genotype and phenotype at the F1/F2 stage (i.e. after breeding) and after getting homozygous mutant animals is highly advisable.14
Reducing animal care costs by making genetic screening more commonplace in biomedical research
Several studies even suggest karyotyping and fully characterizing selected ES cell clones - for the desired genetic modification - prior microinjection, something Cergentis routinely performs for its customers. In the event of gene targeting, it will equally be important to assess that no additional random (transgene) insertions have occurred and accompanied engineering.15 Furthermore, thorough genotypic evaluation of the founder animals should also be carefully performed and interpreted (bearing in mind the possibility of mosaicism).5,10
Given the growing concern and awareness of genotypic mosaicism, it would be prudent and sensible to not only identify and implement robust analytical assay(s) but more importantly, to make in-depth genetic characterization a more common practice. Such precautions can help increase the chance of selecting the founder mice with the highest chance of germline transmissions.12
In conclusion, in-depth genetic screening for the desired mutations prior the creation of genome modified animals can greatly reduce animal care costs and enable the cost-effective management of transgenic facilities by saving valuable time and resources.8,12
World-class partnerships
Thanks to our unmatched genetic insights, we are proud to have established a fruitful and long-lasting relationship with Taconic Biosciences, a leading provider of research models in genetic research. In this application note, Taconic spells out the advantages of our genetic QC solutions over conventional approaches.
References
[1] Gordon, J. W., Scangos, G. A., Plotkin, D. J., Barbosa, J. A. and Ruddle, F. H. (1980). Genetic transformation of mouse embryos by microinjection of purified DNA. Proc. Natl. Acad. Sci. USA 77, 7380-7384.
[2] Palmiter, R. D., Brinster, R. L., Hammer, R. E., Trumbauer, M. E., Rosenfeld, M. G., Birnberg, N. C. and Evans, R. M. (1982a). Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes. Nature 300, 611-615.
[3] Bockamp, E., Sprengel, R., Eshkind, L., Lehmann, T., Braun, J. M., Emmrich, F. and Hengstler, J. G. (2008). Conditional transgenic mouse models: from the basics to genome-wide sets of knockouts and current studies of tissue regeneration. Regen. Med. 3, 217-235.
[4] Gurumurthy CB, Lloyd KCK. Generating mouse models for biomedical research: technological advances. Dis Model Mech. 2019 Jan 8;12(1):dmm029462. doi: 10.1242/dmm.029462. PMID: 30626588; PMCID: PMC6361157.
[5] Taconic Biosciences, Inc. Transgene Mapping Analysis by Targeted Locus Amplification Technology. https://www.taconic.com/pdfs/Transgene-Mapping-Analysis-A4.pdf
[6] Soriano, P. (1999). Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat. Genet. 21, 70-71.
[7] Schilit, S., Ohtsuka, M., Quadros, R. M., & Gurumurthy, C. B. (2016). Pronuclear Injection-Based Targeted Transgenesis. Current protocols in human genetics, 91, 15.10.1–15.10.28. https://doi.org/10.1002/cphg.23
[8] Mehravar M, Shirazi A, Nazari M, Banan M. Mosaicism in CRISPR/Cas9-mediated genome editing. Dev Biol. 2019 Jan 15;445(2):156-162. doi: 10.1016/j.ydbio.2018.10.008. Epub 2018 Oct 22. PMID: 30359560.
[9] Queremel Milani DA, Chauhan PR. Genetics, Mosaicism. [Updated 2021 May 8]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK559193/
[10] Houdebine, L-M. (1997). Genetic mosaicism in the generation of transgenic mice. In CRC Press, Transgenic Animals (233-235).
[11] Mehravar M, Shirazi A, Nazari M, Banan M. Mosaicism in CRISPR/Cas9-mediated genome editing. Dev Biol. 2019 Jan 15;445(2):156-162. doi: 10.1016/j.ydbio.2018.10.008. Epub 2018 Oct 22. PMID: 30359560.
[12] Oliver, D., Yuan, S., McSwiggin, H., & Yan, W. (2015). Pervasive Genotypic Mosaicism in Founder Mice Derived from Genome Editing through Pronuclear Injection. PloS one, 10(6), e0129457. https://doi.org/10.1371/journal.pone.0129457
[13] Hashimoto, M., Yamashita, Y., Takemoto, T., 2016. Electroporation of Cas9 protein/sgRNA into early pronuclear zygotes generates non-mosaic mutants in the mouse. Dev. Biol. 418, 1–9.
[14] Aslan, Y., Tadjuidje, E., Zorn, A.M., Cha, S.-W., 2017. High-efficiency non-mosaic CRISPR-mediated knock-in and indel mutation in F0 Xenopus. Development 144, 2852–2858.
[15] Robinson V, Jennings M. Refinement and Reduction in the Production of Genetically Modified Mice. Alternatives to Laboratory Animals. 2004;32(1_suppl):373-375. doi:10.1177/026119290403201s61
