Generation of pig primary fibroblast cells harboring defective MC4R genes by N-ethyl-N-nitrosourea mutagenesis: A gene-driven, nontransgenic approach to pig improvement

Michiharu Sakurai, Shun-ichi Suzuki, Tadashi Furusawa, Satoshi Mikawa, Tomoyuki Tokunaga, Akira Onishi, Takashi Awata

National Institute of Agrobiological Sciences, Tsukuba, Japan.

DOI: 10.4236/ajmb.2013.33018                

Abstract

Transgenic pigs have been produced with the aim of further improving pigs in terms of economic and environmental traits, but these animals have not been allowed to enter the food chain. As an alternative approach to generating pigs with novel traits of economic importance that cannot be introduced by conventional breeding, we propose a strategy for combining in vitro mutagenesis of pig primary cells with N-ethyl-N-nitrosourea (ENU) and somatic-cell nuclear transfer (SCNT) technology. To explore the feasibility of this strategy, we treated pig primary fibroblast cells with ENU, estimated the per-base mutation frequency induced by the mutagen, clonally cultured about 4000 of the mutagenized cells, and screened them for mutation within the coding region of the melanocortin-4 receptor (MC4R) gene, a key gene in energy homeostasis. Through this screening, we obtained 14 cell clones, each harboring a heterozygous base change within the coding region for MC4R. Of the mutant cell clones, each of two contained a mutant allele encoding MC4R with greatly reduced receptor activity. By SCNT using these cell clones as donors, pigs harboring mutated MC4R alleles with reduced receptor activity can be produced. Our strategy for generating pigs with novel genetic traits likely will be more acceptable to the public than is the use of transgenic technology.

Keywords

Pig; Fibroblasts; N-Ethyl-N-Nitorosourea; Mutagenesis

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Prather, R.S., Shen, M. and Dai, Y. (2008) Genetically modified pigs for medicine and agriculture. Biotechnology and Genetic Engineering Reviews, 25, 245-266.
[2]	Parry, M.A.J., Madgwick, P.J., Bayon, C., et al. (2009) Mutation discovery for crop improvement. Journal of Experimental Botany, 60, 2817-2825. doi:10.1093/jxb/erp189
[3]	Chen, Y., Yee, D., Dains, K., et al. (2000) Genotypebased screen for ENU-induced mutations in the mouse embryonic stem cells. Nature Genetics, 24, 314-317. doi:10.1038/73557
[4]	Chick, W.S., Drechsel, D.A., Hammond, W., et al. (2009) Transmission of mutant phenotypes from ES cells to adult mice. Mammalian Genome, 20, 734-740. doi:10.1007/s00335-009-9228-z
[5]	Greber, B., Lehrach, H. and Himmelbauer, H. (2005) Mouse splice mutant generation from ENU-treated ES cells—A gene-driven approach. Genomics, 85, 557-562. doi:10.1016/j.ygeno.2005.01.011
[6]	Greber, B., Tandara, H., Lehrach, H., et al. (2005) Comparison of PCR-based mutation detection methods and application for identification of mouse Sult1a1 mutant embryonic stem cell clones using pooled templates. Human Mutation, 25, 483-490. doi:10.1002/humu.20168
[7]	Vivian, J.L., Chen, Y., Yee, D., et al. (2002) An allelic series of mutations in Smad2 and Sma4 identified in a genotype-based screen of N-ethyl-N-nitrosourea-mutagenized mouse embryonic stem cells. Proceedings of the National Academy of Sciences of the United States of America, 99, 15542-15547. doi:10.1073/pnas.242474199
[8]	Onishi, A., Iwamoto, M., Akita, T., et al. (2000) Pig cloning by microinjection of fetal fibroblast nuclei. Science, 289, 1188-1190. doi:10.1126/science.289.5482.1188
[9]	Fox, J.L. (2008) Cloned animals deemed safe to eat, but labeling issues loom. Nature Biotechnology, 26, 249-250. doi:10.1038/nbt0308-249
[10]	Tao, Y. (2010) The melanocortin-4 receptor: Physiology, pharmacology, and pathophysiology. Endocrine Reviews, 31, 506-543. doi:10.1210/er.2009-0037
[11]	Huszar, D., Lynch, C.A., Fairchild-Huntress, V., et al. (1997) Targeted disruption of the melanocortin-4 receptor results in obesity in mice. Cell, 88, 131-141. doi:10.1016/S0092-8674(00)81865-6
[12]	Grosse, J. Tarnow, P., Roempler, H., et al. (2006) N-ethyl-N-nitrosourea-based generation of mouse models for mutant G protein-coupled receptors. Physiological Genomics, 26, 209-217. doi:10.1152/physiolgenomics.00289.2005
[13]	Meehan, T.P., Tabeta, K., Du, X., et al. (2006) Point mutations in the melanocortin-4 receptor cause variable obesity in mice. Mammalian Genome, 17, 1162-1171. doi:10.1007/s00335-006-0073-z
[14]	Tao, Y. (2009) Mutations in melanocortin-4 receptor and human obesity. Progress of Molecular Biology and Transitional Science, 88, 173-204.
[15]	Wang, Z. and Tao, Y. (2011) Functional studies on twenty novel naturally occurring melanocortin-4 receptor mutations. Biochimica et Biophysica Acta, 1812, 1190-1199. doi:10.1016/j.bbadis.2011.06.008
[16]	Fan, Z., Sartin, J.L. and Tao, Y. (2008) Pharmacological analyses of two naturally occurring porcine melanocortin4 receptor mutations in domestic pigs. Domestic Animal Endocrinology, 34, 383-390. doi:10.1016/j.domaniend.2007.05.003
[17]	Fan, B., Onteru, S.K., Plastow, G.S., et al. (2009) Detailed characterization of the porcine MC4R gene in relation to fatness and growth. Animal Genetics, 40, 401-409. doi:10.1111/j.1365-2052.2009.01853.x
[18]	Kim, K.S., Larsen, N., Short, T., et al. (2000) A missense variant of the porcine melanocortin-4 receptor (MC4R) gene is associated with fatness, growth, and food intake traits. Mammalian Genome, 11, 131-135. doi:10.1007/s003350010025
[19]	Sembon, S., Suzuki, S., Fuchimoto, D., et al. (2008) Sex identification of pigs using polymerase chain reaction amplification of the amelogenin gene. Zygote, 16, 327-332. doi:10.1017/S0967199408004826
[20]	Ovchinnikov, Y.A., Modyanov, N.N., Broude, N.E., et al. (1986) Pig kidney Na+, K+-ATPase. Primary structure and spacial organization. FEBS Letters, 201, 237-245. doi:10.1016/0014-5793(86)80616-0
[21]	Tao, Y. and Segaloff, D.L. (2003) Functional characterization of melanocortin-4 receptor mutations associated with childhood obesity. Endocrinology, 144, 4544-4551. doi:10.1210/en.2003-0524
[22]	Baker, R.M., Brunette, D.M., Mankovitz, R., et al. (1974) Ouabain-resistant mutants of mouse and hamster cells in culture. Cell, 1, 9-21. doi:10.1016/0092-8674(74)90149-4
[23]	Balachandra Dass, S., Heflich, R.H. and Casciano, D.A. (1997) The mutagenic response at the ouabain resistance locus in T cells of mice exposed to N-ethyl-N-nitrosourea parallels the response at the Hprt locus and correlates with mutation target size. Carcinogenesis, 18, 2233-2237. doi:10.1093/carcin/18.11.2233
[24]	Eldridge, S.R. and Gould, M.N. (1991) Specific locus mutagenesis of human mammary epithelial cells by ultraviolet radiation. International Journal of Radiation Biology, 59, 807-814. doi:10.1080/09553009114550701
[25]	Mankovitz, R., Buchwald, M. and Baker, R.M. (1974) Isolation of ouabain-resistant human diploid cells. Cell, 3, 221-226. doi:10.1016/0092-8674(74)90135-4
[26]	Croyle, M.L., Woo, A.L. and Lingrel, J.B. (1997) Extensive random mutagenesis analysis of the Na+/K+-ATPase subunit identifies known and previously unidentified amino acid residues that alter ouabain sensitivity—Implications for ouabain binding. European Journal of Biochemistry, 248, 488-495. doi:10.1111/j.1432-1033.1997.00488.x
[27]	Monroe, R.J., Bergstrom, R.A., Zheng, et al. (2000) Mouse mutants from chemically mutagenized embryonic stem cells. Nature Genetics, 24, 318-321. doi:10.1038/73563
[28]	Yang, J., Lee, P., Lin, S., et al. (1994) Comparison of mutation spectra induced by N-ethyl-N-nitrosourea in the hprt gene of Mer+ and Mer- diploid human fibroblasts. Carcinogenesis, 15, 959-945. doi:10.1093/carcin/15.5.939
[29]	Yatime, L., Laursen, M., Morth, J.P., et al. (2011) Structural insights into the high affinity binding of cardiotonic steroids to the Na+, K+-ATPase. Journal of Structural Biology, 174, 296-306. doi:10.1016/j.jsb.2010.12.004
[30]	Takahasi, K.R., Sakuraba, Y. and Gondo, Y. (2007) Mutational pattern and frequency of induced nucleotide changes in mouse ENU mutagenesis. BMC Molecular Biology, 8, 52-61. doi:10.1186/1471-2199-8-52
[31]	Lingrel, J.B. (2010) The physiological significance of the cardiotonic steroid/ouabain-binding site of the Na, K-ATPase. Annual Reviews of Physiology, 72, 395-412. doi:10.1146/annurev-physiol-021909-135725
[32]	Giuffra, E., Kijas, J.M.H., Amarger, V., et al. (2000) The origin of the domestic pig: Independent domestication and subsequent introgression. Genetics, 154, 1785-1791.
[33]	Markel, P., Shu, P., Ebeling, C., et al. (1997) Theoretical and empirical issues for marker-assisted breeding of congenic mouse strains. Nature Genetics, 17, 280-284. doi:10.1038/ng1197-280
[34]	Groenen, M.A.M., Archibald, A.L., Uenishi, H., et al. (2012) Analyses of pig genomes provide insight into porcine demography and evolution. Nature, 491, 393-398. doi:10.1038/nature11622
[35]	Ramos, A.M., Crooijmans, R.P.M.A., Affara, N.A., et al. (2011) Design of high density SNP genotyping assay in the pig using SNPs identified and characterized by next generation sequencing technology. PLoS ONE, 4, e6524. http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0006524 doi:10.1371/journal.pone.0006524
[36]	Carlson, D.F., Tan, W., Lillico, S.G., et al. (2012) Efficient TALEN-mediated gene knockout in livestock. Proceedings of the National Academy of Sciences of the United States of America, 109, 17382-17387. doi:10.1073/pnas.1211446109
[37]	Yang, D., Yang, H., Li, W., et al. (2011) Generation of PPAR mono-allelic knockout pigs via zinc-finger nucleases and nuclear transfer cloning. Cell Research, 21, 979-982. doi:10.1038/cr.2011.70
[38]	Chandrasekharan, S., Kumar, S., Valley, C.M., et al. (2009) Proprietary science, open science and the role of patent disclosure: The case of zinc-finger proteins. Nature Biotechnology, 27, 140-144. doi:10.1038/nbt0209-140
[39]	DeFrancesco, L. (2011) More over ZFNs. Nature Biotechnology, 29, 681-684. doi:10.1038/nbt.1935