DOI QR코드

DOI QR Code

Myotube differentiation in clustered regularly interspaced short palindromic repeat/Cas9-mediated MyoD knockout quail myoblast cells

  • Kim, Si Won ;
  • Lee, Jeong Hyo ;
  • Park, Byung-Chul ;
  • Park, Tae Sub
  • Received : 2016.09.29
  • Accepted : 2016.10.24
  • Published : 2017.07.01

Abstract

Objective: In the livestock industry, the regulatory mechanisms of muscle proliferation and differentiation can be applied to improve traits such as growth and meat production. We investigated the regulatory pathway of MyoD and its role in muscle differentiation in quail myoblast cells. Methods: The MyoD gene was mutated by the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology and single cell-derived MyoD mutant sublines were identified to investigate the global regulatory mechanism responsible for muscle differentiation. Results: The mutation efficiency was 73.3% in the mixed population, and from this population we were able to establish two QM7 MyoD knockout subline (MyoD KO QM7#4) through single cell pick-up and expansion. In the undifferentiated condition, paired box 7 expression in MyoD KO QM7#4 cells was not significantly different from regular QM7 (rQM7) cells. During differentiation, however, myotube formation was dramatically repressed in MyoD KO QM7#4 cells. Moreover, myogenic differentiation-specific transcripts and proteins were not expressed in MyoD KO QM7#4 cells even after an extended differentiation period. These results indicate that MyoD is critical for muscle differentiation. Furthermore, we analyzed the global regulatory interactions by RNA sequencing during muscle differentiation. Conclusion: With CRISPR/Cas9-mediated genomic editing, single cell-derived sublines with a specific knockout gene can be adapted to various aspects of basic research as well as in functional genomics studies.

Keywords

Myoblast;CRISPR-Cas9;Knockout;Muscle Differentiation;MyoD

References

  1. Hillier LW, Miller W, Birney E et al. Sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution. Nature 2004;432:695-716. https://doi.org/10.1038/nature03154
  2. Rubin CJ, Zody MC, Eriksson J, et al. Whole-genome resequencing reveals loci under selection during chicken domestication. Nature 2010;464:587-91. https://doi.org/10.1038/nature08832
  3. Al-Musawi SL, Lock F, Simbi BH, Bayol SAM, Stickland NC. Muscle specific differences in the regulation of myogenic differentiation in chickens genetically selected for divergent growth rates. Differentiation 2011;82:127-35. https://doi.org/10.1016/j.diff.2011.05.012
  4. Shin S, Song Y, Ahn J, et al. A novel mechanism of myostatin regulation by its alternative splicing variant during myogenesis in avian species. Am J Physiol Cell Physiol 2015;309:650-9. https://doi.org/10.1152/ajpcell.00187.2015
  5. Park TS, Han JY. Genetic modification of chicken germ cells. Ann NY Acad Sci 2012;1271:104-9. https://doi.org/10.1111/j.1749-6632.2012.06744.x
  6. Park TS, Han JY. piggyBac transposition into primordial germ cells is an efficient tool for transgenesis in chickens. Proc Natl Acad Sci USA 2012;109:9337-41. https://doi.org/10.1073/pnas.1203823109
  7. Park TS, Lee HJ, Kim KH, Kim JS, Han JY. Targeted gene knockout in chickens mediated by TALENs. Proc Natl Acad Sci USA 2014;111:12716-21. https://doi.org/10.1073/pnas.1410555111
  8. Salter DW, Smith EJ, Hughes SH, et al. Gene insertion into the chicken germ line by retroviruses. Poult Sci 1986;65:1445-8. https://doi.org/10.3382/ps.0651445
  9. Love J, Gribbin C, Mather C, Sang H. Transgenic birds by DNA microinjection. Biotechnology 1994;12:60-3.
  10. Macdonald J, Taylor L, Sherman A, et al. Efficient genetic modification and germ-line transmission of primordial germ cells using piggyBac and Tol2 transposons. Proc Natl Acad Sci USA 2012;109: E1466-E72. https://doi.org/10.1073/pnas.1118715109
  11. Schusser B, Collarini EJ, Yi H, et al. Immunoglobulin knockout chickens via efficient homologous recombination in primordial germ cells. Proc Natl Acad Sci USA 2013;110:20170-5. https://doi.org/10.1073/pnas.1317106110
  12. Dimitrov L, Pedersen D, Ching KH, et al. Germline gene editing in chickens by efficient CRISPR-mediated homologous recombination in primordial germ cells. PLoS ONE 2016;11:e0154303. https://doi.org/10.1371/journal.pone.0154303
  13. Cong L, Ran FA, Cox D, et al. Multiplex genome engineering using CRISPR/Cas systems. Science 2013;339:819-23. https://doi.org/10.1126/science.1231143
  14. Mali P, Yang L, Esvelt KM, et al. RNA-guided human genome engineering via Cas9. Science 2013;339:823-6. https://doi.org/10.1126/science.1232033
  15. Davis RL, Weintraub H, Lassar AB. Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell 1987;51:987-1000. https://doi.org/10.1016/0092-8674(87)90585-X
  16. Rudnicki MA, Schnegelsberg PNJ, Stead RH, et al. MyoD or Myf-5 is required for the formation of skeletal muscle. Cell 1993;75:1351-9. https://doi.org/10.1016/0092-8674(93)90621-V
  17. Berkes CA, Tapscott SJ. MyoD and the transcriptional control of myogenesis. Semin Cell Dev Biol 2005;16:585-95. https://doi.org/10.1016/j.semcdb.2005.07.006
  18. Buckingham M. Myogenic progenitor cells and skeletal myogenesis in vertebrates. Curr Opin Genet Dev 2006;16:525-32. https://doi.org/10.1016/j.gde.2006.08.008
  19. Rudnicki MA, Braun T, Hinuma S, Jaenisch R. Inactivation of MyoD in mice leads to up-regulation of the myogenic HLH gene Myf-5 and results in apparently normal muscle development. Cell 1992;71:383-90. https://doi.org/10.1016/0092-8674(92)90508-A
  20. Arnold HH, Braun T. Targeted inactivation of myogenic factor genes reveals their role during mouse myogenesis: a review. Int J Dev Biol 1996;40:345-53.
  21. Wang H, Yang H, Shivalila CS, et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 2013;153:910-8. https://doi.org/10.1016/j.cell.2013.04.025
  22. Jao LE, Wente SR, Chen W. Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proc Natl Acad Sci USA 2013;110:13904-9. https://doi.org/10.1073/pnas.1308335110
  23. Hai T, Teng F, Guo R, Li W, Zhou Q. One-step generation of knockout pigs by zygote injection of CRISPR/Cas system. Cell Res 2014;24:372-5. https://doi.org/10.1038/cr.2014.11
  24. Weber D, Wiese C, Gessler M. Hey bHLH transcription factors. Curr Top Dev Biol 2014;110:285-315.
  25. Benezra R, Davis RL, Lockshon D, Turner DL, Weintraub H. The protein Id: a negative regulator of helix-loop-helix DNA binding proteins. Cell 1990;61:49-59. https://doi.org/10.1016/0092-8674(90)90214-Y
  26. Ruzinova MB, Benezra R. Id proteins in development, cell cycle and cancer. Trends Cell Biol 2003;13:410-8. https://doi.org/10.1016/S0962-8924(03)00147-8
  27. Perk J, Iavarone A, Benezra R. The Id family of helix-loop-helix proteins in cancer. Nat Rev Cancer 2005;5:603-14. https://doi.org/10.1038/nrc1673
  28. Kaplan FS, Xu M, Seemann P, et al. Classic and atypical fibrodysplasia ossificans progressiva (FOP) phenotypes are caused by mutations in the bone morphogenetic protein (BMP) type I receptor ACVR1. Hum Mutat 2009;30:379-90. https://doi.org/10.1002/humu.20868

Cited by

  1. CRISPR-Trap: a clean approach for the generation of gene knockouts and gene replacements in human cells vol.29, pp.2, 2018, https://doi.org/10.1091/mbc.E17-05-0288
  2. Muscle differentiation induced up-regulation of calcium-related gene expression in quail myoblasts vol.31, pp.9, 2018, https://doi.org/10.5713/ajas.18.0302
  3. Forkhead box O3 promotes cell proliferation and inhibits myotube differentiation in chicken myoblast cells vol.60, pp.1, 2019, https://doi.org/10.1080/00071668.2018.1547362

Acknowledgement

Grant : Cooperative Research Program for Agriculture Science & Technology Developmen

Supported by : Rural Development Administration, Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET)