Abstract
Ability to disrupt genes is essential in elucidating gene function. Unlike rodents or amphibians, it has been difficult to generate gene-targeted embryos in large animals. Therefore, studies of early embryo development have been hampered in large animals. A recent technology suggests that targeted mutations can be successfully introduced during embryogenesis, thus by-passing the need of breeding to produce gene-targeted embryos. This is particularly important in large animal models because of longer gestation period and higher animal cost. Here, we describe a specific approach to disrupt up to two genes simultaneously during embryogenesis using the CRISPR/Cas9 technology in swine. The approach can help understand the mechanism of zygotic genome activation in large animals.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Capecchi MR (2005) Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century. Nat Rev Genet 6(6):507–512. doi:10.1038/nrg1619
Thomas KR, Capecchi MR (1987) Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51(3):503–512
Wilke M, Buijs-Offerman RM, Aarbiou J, Colledge WH, Sheppard DN, Touqui L, Bot A, Jorna H, de Jonge HR, Scholte BJ (2011) Mouse models of cystic fibrosis: phenotypic analysis and research applications. J Cyst Fibros 10(Suppl 2):S152–S171. doi:10.1016/s1569-1993(11)60020-9
Braude P, Bolton V, Moore S (1988) Human gene expression first occurs between the four- and eight-cell stages of preimplantation development. Nature 332(6163):459–461. doi:10.1038/332459a0
Prather RS (1993) Nuclear control of early embryonic development in domestic pigs. J Reprod Fertil Suppl 48:17–29
Santos F, Hyslop L, Stojkovic P, Leary C, Murdoch A, Reik W, Stojkovic M, Herbert M, Dean W (2010) Evaluation of epigenetic marks in human embryos derived from IVF and ICSI. Hum Reprod 25(9):2387–2395. doi:10.1093/humrep/deq151
Deshmukh RS, Ostrup O, Ostrup E, Vejlsted M, Niemann H, Lucas-Hahn A, Petersen B, Li J, Callesen H, Hyttel P (2011) DNA methylation in porcine preimplantation embryos developed in vivo and produced by in vitro fertilization, parthenogenetic activation and somatic cell nuclear transfer. Epigenetics 6(2):177–187
Lai L, Kolber-Simonds D, Park KW, Cheong HT, Greenstein JL, Im GS, Samuel M, Bonk A, Rieke A, Day BN, Murphy CN, Carter DB, Hawley RJ, Prather RS (2002) Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science (New York, NY) 295(5557):1089–1092. doi:10.1126/science.1068228
Dai Y, Vaught TD, Boone J, Chen SH, Phelps CJ, Ball S, Monahan JA, Jobst PM, McCreath KJ, Lamborn AE, Cowell-Lucero JL, Wells KD, Colman A, Polejaeva IA, Ayares DL (2002) Targeted disruption of the alpha1,3-galactosyltransferase gene in cloned pigs. Nat Biotechnol 20(3):251–255. doi:10.1038/nbt0302-251
Whitworth KM, Lee K, Benne JA, Beaton BP, Spate LD, Murphy SL, Samuel MS, Mao J, O'Gorman C, Walters EM, Murphy CN, Driver JP, Mileham A, McLaren D, Wells KD, Prather RS (2014) Use of the CRISPR/Cas9 system to produce genetically engineered pigs from in vitro-derived oocytes and embryos. Biol Reprod 91:78. doi:10.1095/biolreprod.114.121723
Lei S, Ryu J, Wen K, Twitchell E, Bui T, Ramesh A, Weiss M, Li G, Samuel H, Clark-Deener S, Jiang X, Lee K, Yuan L (2016) Increased and prolonged human norovirus infection in RAG2/IL2RG deficient gnotobiotic pigs with severe combined immunodeficiency. Sci Rep 6:25222. doi:10.1038/srep25222
Yoshioka K, Suzuki C, Tanaka A, Anas IM, Iwamura S (2002) Birth of piglets derived from porcine zygotes cultured in a chemically defined medium. Biol Reprod 66(1):112–119
Abeydeera LR, Wang WH, Cantley TC, Rieke A, Prather RS, Day BN (1998) Presence of epidermal growth factor during in vitro maturation of pig oocytes and embryo culture can modulate blastocyst development after in vitro fertilization. Mol Reprod Dev 51(4):395–401. doi:10.1002/(sici)1098-2795(199812)51:4<395::aid-mrd6>3.0.co;2-y
Lee K, Redel BK, Spate L, Teson J, Brown AN, Park KW, Walters E, Samuel M, Murphy CN, Prather RS (2013) Piglets produced from cloned blastocysts cultured in vitro with GM-CSF. Mol Reprod Dev 80(2):145–154. doi:10.1002/mrd.22143
Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R (2013) One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153(4):910–918. doi:10.1016/j.cell.2013.04.025
Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8(11):2281–2308. doi:10.1038/nprot.2013.143
Acknowledgment
This work was supported by NIH grant R21OD019934.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Ryu, J., Lee, K. (2017). CRISPR/Cas9-Mediated Gene Targeting during Embryogenesis in Swine. In: Lee, K. (eds) Zygotic Genome Activation. Methods in Molecular Biology, vol 1605. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6988-3_16
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6988-3_16
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6986-9
Online ISBN: 978-1-4939-6988-3
eBook Packages: Springer Protocols