Adapting Mouse Genome Editing Technique from Scratch Using in utero Electroporation

Cover Page

Cite item

Full Text

Open Access Open Access
Restricted Access Access granted
Restricted Access Subscription Access

Abstract

Mouse genome modification requires costly equipment and highly skilled personnel to manipulate zygotes. A number of zygote electroporation techniques were reported to be highly efficient in gene delivery. One of these methods called i-GONAD (improved Genome-editing via Oviductal Nucleic Acids Delivery) describes electroporation-based gene transfer to zygotes in utero. Here we adopted this technology to develop an easy-to-use and cost-effective pipeline enabling mouse genome-editing from scratch with minimal requirements to operator skills and animal use. We chose the CRISPR/Cas9 system as a genome editing tool and i-GONAD as a gene delivery method to produce Il10 knockout in C57BL/6 mice. Three animals out of 13 delivered pups (23%) were genetically compromised at Il10 locus suggesting the feasibility of the approach. This protocol provides detailed description of the used technical settings paired with troubleshooting tips and could be of interest to those who aim at establishing in-house mouse transgenesis pipeline at minimal equipment cost from scratch.

Full Text

Restricted Access

About the authors

Yu. V. Popova

Institute of Molecular and Cellular Biology of the Siberian Branch of the Russian Academy of Sciences; Novosibirsk State Agrarian University

Email: kozhevnikova@mcb.nsc.ru
Russian Federation, Novosibirsk, 630090; Novosibirsk, 630039

V. D. Bets

Novosibirsk State Technical University

Email: kozhevnikova@mcb.nsc.ru
Russian Federation, Novosibirsk, 630073

E. S. Omelina

Institute of Molecular and Cellular Biology of the Siberian Branch of the Russian Academy of Sciences

Email: kozhevnikova@mcb.nsc.ru
Russian Federation, Novosibirsk, 630090

L. V. Boldyreva

Institute of Molecular and Cellular Biology of the Siberian Branch of the Russian Academy of Sciences; Scientific Research Institute of Neurosciences and Medicine, Novosibirsk, 630117

Email: kozhevnikova@mcb.nsc.ru
Russian Federation, Novosibirsk, 630090; Novosibirsk, 630117

E. N. Kozhevnikova

Institute of Molecular and Cellular Biology of the Siberian Branch of the Russian Academy of Sciences; Novosibirsk State Agrarian University

Author for correspondence.
Email: kozhevnikova@mcb.nsc.ru
Russian Federation, Novosibirsk, 630090; Novosibirsk, 630039

References

  1. Takahashi G., Gurumurthy C.B., Wada K., Miura H., Sato M., Ohtsuka M. (2015) GONAD: Genome-editing via oviductal nucleic acids delivery system: a novel microinjection independent genome engineering method in mice. Sci. Rep. 5, 11406. https://doi.org/10.1038/srep11406
  2. Ohtsuka M., Sato M., Miura H., Takabayashi S., Matsuyama M., Koyano T., Arifin N., Nakamura S., Wada K., Gurumurthy C.B. (2018) I-GONAD: A robust method for in situ germline genome engineering using CRISPR nucleases. Genome Biol. 19, 25. https://doi.org/10.1186/s13059-018-1400-x
  3. Sato M., Nakamura S., Inada E., Takabayashi S. (2022) Recent advances in the production of genome-edited rats. Int. J. Mol. Sci. 23(5), 2548. https://doi.org/10.3390/ijms23052548
  4. Hirose M., Tomishima T., Ogura A. (2023) Editing the genome of the golden hamster (Mesocricetus auratus). Meth. Mol. Biol. 2637, 247–254. https://doi.org/10.1007/978-1-0716-3016-7_19
  5. Namba M., Kobayashi T., Koyano T., Kohno M., Ohtsuka M., Matsuyama M. (2021) GONAD: A new method for germline genome editing in mice and rats. Dev. Growth. Differ. 63(8), 439–447. https://doi.org/10.1111/dgd.12746
  6. Kobayashi Y., Aoshima T., Ito R., Shinmura R., Ohtsuka M., Akasaka E., Sato M., Takabayashi S. (2020) Modification of i-GONAD suitable for production of genome-edited C57BL/6 inbred mouse strain. Cells. 9(4), 957. https://doi.org/10.3390/cells9040957
  7. Shang R., Zhang H., Bi P. (2021) Generation of mouse conditional knockout alleles in one step using the i-GONAD method. Gen. Res. 31(1), 121–130. https://doi.org/10.1101/gr.265439.120
  8. Sato M., Nakamura A., Sekiguchi M., Matsuwaki T., Miura H., Gurumurthy C.B., Kakuta S., Ohtsuka M. (2023) Improved genome editing via oviductal nucleic acids delivery (i-GONAD): Protocol steps and additional notes. Meth. Mol. Biol. 2631, 325–340. https://doi.org/10.1007/978-1-0716-2990-1_14
  9. Melo-Silva C.R., Knudson C.J., Tang L., Kafle S., Springer L.E., Choi J., Snyder C.M., Wang Y., Kim S.V., Sigal L.J. (2023) Multiple and consecutive genome editing using i-GONAD and breeding enrichment facilitates the production of genetically modified mice. Cells. 12(9), 1343. https://doi.org/10.3390/cells12091343
  10. Gurumurthy C.B., Sato M., Nakamura A., Inui M., Kawano N., Islam M.A., Ogiwara S., Takabayashi S., Matsuyama M., Nakagawa S., Miura H., Ohtsuka M. (2019) Creation of CRISPR-based germline-genome-engineered mice without ex vivo handling of zygotes by i-GONAD. Nat. Protoc. 14(8), 2452–2482. https://doi.org/10.1038/s41596-019-0187-x
  11. Garcia-Frigola C., Carreres M.I., Vegar C., Herrera E. (2007) Gene delivery into mouse retinal ganglion cells by in utero electroporation. BMC Dev. Biol. 7, 103. https://doi.org/10.1186/1471-213X-7-103
  12. Shinmyo Y., Tanaka S., Tsunoda S., Hosomichi K., Tajima A., Kawasaki H. (2016) CRISPR/Cas9-mediated gene knockout in the mouse brain using in utero electroporation. Sci. Rep. 6, 20611. https://doi.org/10.1038/srep20611
  13. Book Reviews. (2002) J. Vet. Med. Educ. 29, 245–246. https://doi.org/10.3138/jvme.29.4.245
  14. Cagle L.A., Franzi L.M., Epstein S.E., Kass P.H., Last J.A., Kenyon N.J. (2017) Injectable anesthesia for mice: combined effects of dexmedetomidine, tiletamine-zolazepam, and butorphanol. Anesthesiol. Res. Pract. 2017, 9161040. https://doi.org/10.1155/2017/9161040
  15. Limprasutr V., Sharp P., Jampachaisri K., Pacharinsak C., Durongphongtorn S. (2021) Tiletamine/zolazepam and dexmedetomidine with tramadol provide effective general anesthesia in rats. Animal Model Exp. Med. 4(1), 40–46. https://doi.org/10.1002/ame2.12143
  16. Cohen J. (2016) “Any idiot can do it.” Genome editor CRISPR could put mutant mice in everyone’s reach. Science. https://doi.org/10.1126/science.aal0334
  17. Modzelewski A.J., Chen S., Willis B.J., Lloyd K.C.K., Wood J.A., He L. (2018) Efficient mouse genome engineering by CRISPR-EZ technology. Nat. Protoc. 13(6), 1253–1274. https://doi.org/10.1038/nprot.2018.012
  18. Imai Y., Tanave A., Matsuyama M., Koide T. (2022) Efficient genome editing in wild strains of mice using the i-GONAD method. Sci. Rep. 12(1), 13821. https://doi.org/10.1038/s41598-022-17776-x
  19. Weber E.M., Algers B., Würbel H., Hultgren J., Olsson I.A.S. (2013) Influence of strain and parity on the risk of litter loss in laboratory mice. Reprod. Domest Anim. 48(2), 292–296. https://doi.org/10.1111/j.1439-0531.2012.02147.x
  20. Carter D.B., Kennett M.J., Franklin C.L. (2002) Use of perphenazine to control cannibalism in DBA/1 mice. Comp. Med. 52(5), 452–455.
  21. Du Sert N.P., Hurst V., Ahluwalia A., Alam S., Avey M.T., Baker M., Browne W.J., Clark A., Cuthill I.C., Dirnagl U., Emerson M., Garner P., Holgate S.T., Howells D.W., Karp N.A., Lazic S.E., Lidster K., MacCallum C.J., Macleod M., Pearl E.J., Petersen O.H., Rawle F., Reynolds P., Rooney K., Sena E.S., Silberberg S.D., Steckler T., Würbel H. (2020) The arrive guidelines 2.0: Updated guidelines for reporting animal research. PLoS Biol. 18(7), e3000410. https://doi.org/10.1371/journal.pbio.3000410

Supplementary files

Supplementary Files
Action
1. JATS XML
Download
2. Appendix
Download (1MB)
Indexing metadata
3. Fig. 1. The scheme of CRISPR/Cas9 genome editing in combination with the i-GONAD method. a – Synthesis of sgRNA (single guide RNA). b – The i-GONAD method (improved Genome-editing via Oviductal Nucleic Acids Delivery). b – Suturing and postoperative procedures. The drawing was created on the website biorender.com.

Download (713KB)
Indexing metadata
4. Fig. 2. Adaptation and testing of the i-GONAD method. a – Testing of sdRNA in vitro. Electrophoresis in agarose gel of a ScaI-linearized pBluescript vector SK (+) carrying a fragment of the genomic region of exon 1 or exon 2 of the Il10 gene. RNP – ribonuclear complexes of cdRNA and Cas9 protein. b – Survival of zygotes in vitro after electroporation in utero. EP – electroporation. b – In utero electroporation efficiency test using 4kDa FITC-dextran (FD4).

Download (571KB)
Indexing metadata
5. Fig. 3. Genotyping of offspring. a is a schematic illustration of the wild–type Il10 locus. Exons (from x1 to x5) and introns are indicated by black rectangles and a gray line, respectively. The two target sequences (ex1 and ex2) are underlined and shown in blue. The sequences of the motif adjacent to the protospacer (PAM) are shown in red. b – Direct Sanger sequencing of the genomic regions of the Il10 gene in F1 progeny. Transgene #58 has abnormalities in exon 1, the other two (#27 and #32) in exon 2 of the Il10 gene. b – Comparison of the amino acid sequences of the wild-type Il10 gene and offspring after i-GONAD. The control amino acid sequence of the wild-type Il10 protein is shown above. The amino acid sequences of the mutated Il10 proteins (derived from transgenes #58, #27 and #32) are shown below. The predicted consequences of mutation of amino acid sequences are highlighted in red. The stop codons are marked with *.

Download (1MB)
Indexing metadata

Copyright (c) 2024 Russian Academy of Sciences