Laboraroty Animal Research

Korean Association for Laboratory Animal Science (한국실험동물학회)
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CRISPR/Cas9-mediated generation of a Plac8 knockout mouse model

  • Lee, HyunJeong (Department of Biochemistry, College of Life Science and Biotechnology and Yonsei Laboratory Animal Research Center, Yonsei University) ;
  • Kim, Joo-Il (Graduate School of Translational Medicine, Seoul National University College of Medicine) ;
  • Park, Jin-Sung (Department of Experimental Animal Research, Biomedical Research Institute, Seoul National Univ. Hospital) ;
  • Roh, Jae-il (Department of Biochemistry, College of Life Science and Biotechnology and Yonsei Laboratory Animal Research Center, Yonsei University) ;
  • Lee, Jaehoon (Department of Biochemistry, College of Life Science and Biotechnology and Yonsei Laboratory Animal Research Center, Yonsei University) ;
  • Kang, Byeong-Cheol (Graduate School of Translational Medicine, Seoul National University College of Medicine) ;
  • Lee, Han-Woong (Department of Biochemistry, College of Life Science and Biotechnology and Yonsei Laboratory Animal Research Center, Yonsei University)
  • Received : 2018.11.15
  • Accepted : 2018.12.08
  • Published : 2018.12.31
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Abstract

Placenta specific 8 (PLAC8, also known as ONZIN) is a multi-functional protein that is highly expressed in the intestine, lung, spleen, and innate immune cells, and is involved in various diseases, including cancers, obesity, and innate immune deficiency. Here, we generated a Plac8 knockout mouse using the CRISPR/Cas9 system. The Cas9 mRNA and two single guide RNAs targeting a region near the translation start codon at Plac8 exon 2 were microinjected into mouse zygotes. This successfully eliminated the conventional translation start site, as confirmed by Sanger sequencing and PCR genotyping analysis. Unlike the previous Plac8 deficient models displaying increased adipose tissue and body weights, our male Plac8 knockout mice showed rather lower body weight than sex-matched littermate controls, though the only difference between these two mouse models is genetic context. Differently from the previously constructed embryonic stem cell-derived Plac8 knockout mouse that contains a neomycin resistance cassette, this knockout mouse model is free from a negative selection marker or other external insertions, which will be useful in future studies aimed at elucidating the multi-functional and physiological roles of PLAC8 in various diseases, without interference from exogenous foreign DNA.

Keywords

Acknowledgement

Supported by : National Research Foundation of the Republic of Korea, Korea's Ministry of Food and Drug Safety (MFDS)

References

  1. Artelt P, Grannemann R, Stocking C, Friel J, Bartsch J, Hauser H. The prokaryotic neomycin-resistance-encoding gene acts as a transcriptional silencer in eukaryotic cells. Gene 1991; 99(2): 249-254. https://doi.org/10.1016/0378-1119(91)90134-W
  2. Biankin AV, Waddell N, Kassahn KS, Gingras MC, Muthuswamy LB, Johns AL, Miller DK, Wilson PJ, Patch AM, Wu J, Chang DK, Cowley MJ, Gardiner BB, Song S, Harliwong I, Idrisoglu S, Nourse C, Nourbakhsh E, Manning S, Wani S, Gongora M, Pajic M, Scarlett CJ, Gill AJ, Pinho AV, Rooman I, Anderson M, Holmes O, Leonard C, Taylor D, Wood S, Xu Q, Nones K, Fink JL, Christ A, Bruxner T, Cloonan N, Kolle G, Newell F, Pinese M, Mead RS, Humphris JL, Kaplan W, Jones MD, Colvin EK, Nagrial AM, Humphrey ES, Chou A, Chin VT, Chantrill LA, Mawson A, Samra JS, Kench JG, Lovell JA, Daly RJ, Merrett ND, Toon C, Epari K, Nguyen NQ, Barbour A, Zeps N; Australian Pancreatic Cancer Genome Initiative, Kakkar N, Zhao F, Wu YQ, Wang M, Muzny DM, Fisher WE, Brunicardi FC, Hodges SE, Reid JG, Drummond J, Chang K, Han Y, Lewis LR, Dinh H, Buhay CJ, Beck T, Timms L, Sam M, Begley K, Brown A, Pai D, Panchal A, Buchner N, De Borja R, Denroche RE, Yung CK, Serra S, Onetto N, Mukhopadhyay D, Tsao MS, Shaw PA, Petersen GM, Gallinger S, Hruban RH, Maitra A, Iacobuzio- Donahue CA, Schulick RD, Wolfgang CL, Morgan RA, Lawlor RT, Capelli P, Corbo V, Scardoni M, Tortora G, Tempero MA, Mann KM, Jenkins NA, Perez-Mancera PA, Adams DJ, Largaespada DA, Wessels LF, Rust AG, Stein LD, Tuveson DA, Copeland NG, Musgrove EA, Scarpa A, Eshleman JR, Hudson TJ, Sutherland RL, Wheeler DA, Pearson JV, McPherson JD, Gibbs RA, Grimmond SM. Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes. Nature 2012; 491(7424): 399-405. https://doi.org/10.1038/nature11547
  3. Brabletz T, Hlubek F, Spaderna S, Schmalhofer O, Hiendlmeyer E, Jung A, Kirchner T. Invasion and metastasis in colorectal cancer: epithelial-mesenchymal transition, mesenchymal-epithelial transition, stem cells and beta-catenin. Cells Tissues Organs 2005; 179(1-2): 56-65. https://doi.org/10.1159/000084509
  4. Brouns SJ, Jore MM, Lundgren M, Westra ER, Slijkhuis RJ, Snijders AP, Dickman MJ, Makarova KS, Koonin EV, van der Oost J. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 2008; 321(5891): 960-964. https://doi.org/10.1126/science.1159689
  5. Choi JY, Yun WB, Kim JE, Lee MR, Park JJ, Song BR, Kim HR, Park JW, Kang MJ, Kang BC, Lee HW, Hwang DY. Successful development of squamous cell carcinoma and hyperplasia in RGEN-mediated p27 KO mice after the treatment of DMBA and TPA. Lab Anim Res 2018; 34(3): 118-125. https://doi.org/10.5625/lar.2018.34.3.118
  6. Choy MC, Visvanathan K, De Cruz P. An Overview of the Innate and Adaptive Immune System in Inflammatory Bowel Disease. Inflamm Bowel Dis 2017; 23(1): 2-13. https://doi.org/10.1097/MIB.0000000000000955
  7. Couillard C, Mauriege P, Imbeault P, Prud'homme D, Nadeau A, Tremblay A, Bouchard C, Despres JP. Hyperleptinemia is more closely associated with adipose cell hypertrophy than with adipose tissue hyperplasia. Int J Obes Relat Metab Disord 2000; 24(6): 782-788. https://doi.org/10.1038/sj.ijo.0801227
  8. Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW, Donovan KF, Smith I, Tothova Z, Wilen C, Orchard R, Virgin HW, Listgarten J, Root DE. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol 2016; 34(2): 184-191. https://doi.org/10.1038/nbt.3437
  9. Galaviz-Hernandez C, Stagg C, de Ridder G, Tanaka TS, Ko MS, Schlessinger D, Nagaraja R. Plac8 and Plac9, novel placentalenriched genes identified through microarray analysis. Gene 2003; 309(2): 81-89. https://doi.org/10.1016/S0378-1119(03)00508-0
  10. Gukovsky I, Li N, Todoric J, Gukovskaya A, Karin M. Inflammation, autophagy, and obesity: common features in the pathogenesis of pancreatitis and pancreatic cancer. Gastroenterology 2013; 144(6): 1199-1209. https://doi.org/10.1053/j.gastro.2013.02.007
  11. Hickey MJ, Kubes P. Intravascular immunity: the host-pathogen encounter in blood vessels. Nat Rev Immunol 2009; 9(5): 364-375. https://doi.org/10.1038/nri2532
  12. Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, Cradick TJ, Marraffini LA, Bao G, Zhang F. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 2013; 31(9): 827-832. https://doi.org/10.1038/nbt.2647
  13. Jimenez-Preitner M, Berney X, Thorens B. Plac8 is required for white adipocyte differentiation in vitro and cell number control in vivo. PLoS One 2012; 7(11): e48767. https://doi.org/10.1371/journal.pone.0048767
  14. Jimenez-Preitner M, Berney X, Uldry M, Vitali A, Cinti S, Ledford JG, Thorens B. Plac8 is an inducer of $C/EBP{\beta}$ required for brown fat differentiation, thermoregulation, and control of body weight. Cell Metab 2011; 14(5): 658-670. https://doi.org/10.1016/j.cmet.2011.08.008
  15. Jimenez-Preitner M, Berney X, Uldry M, Vitali A, Cinti S, Ledford JG, Thorens B. Plac8 is an inducer of $C/EBP{\beta}$ required for brown fat differentiation, thermoregulation, and control of body weight. Cell Metab 2011; 14(5): 658-670. https://doi.org/10.1016/j.cmet.2011.08.008
  16. Kaistha BP, Lorenz H, Schmidt H, Sipos B, Pawlak M, Gierke B, Kreider R, Lankat-Buttgereit B, Sauer M, Fiedler L, Krattenmacher A, Geisel B, Kraus JM, Frese KK, Kelkenberg S, Giese NA, Kestler HA, Gress TM, Buchholz M. PLAC8 Localizes to the Inner Plasma Membrane of Pancreatic Cancer Cells and Regulates Cell Growth and Disease Progression through Critical Cell-Cycle Regulatory Pathways. Cancer Res 2016; 76(1): 96-107. https://doi.org/10.1158/0008-5472.CAN-15-0216
  17. Kang Y, Massague J. Epithelial-mesenchymal transitions: twist in development and metastasis. Cell 2004; 118(3): 277-279. https://doi.org/10.1016/j.cell.2004.07.011
  18. Kim JH, Cho EY, Kwon E, Kim WH, Park JS, Lee YS, Yun JW, Kang BC. Gold thread implantation promotes hair growth in human and mice. Lab Anim Res 2017; 33(4): 291-297. https://doi.org/10.5625/lar.2017.33.4.291
  19. Kinsey C, Balakrishnan V, O'Dell MR, Huang JL, Newman L, Whitney-Miller CL, Hezel AF, Land H. Plac8 links oncogenic mutations to regulation of autophagy and is critical to pancreatic cancer progression. Cell Rep 2014; 7(4): 1143-1155. https://doi.org/10.1016/j.celrep.2014.03.061
  20. Krzizek EC, Brix JM, Herz CT, Kopp HP, Schernthaner GH, Schernthaner G, Ludvik B. Prevalence of Micronutrient Deficiency in Patients with Morbid Obesity Before Bariatric Surgery. Obes Surg 2018; 28(3): 643-648. https://doi.org/10.1007/s11695-017-2902-4
  21. Lachmann PJ. Microbial subversion of the immune response. Proc Natl Acad Sci U S A 2002; 99(13): 8461-8462. https://doi.org/10.1073/pnas.132284499
  22. Ledford JG, Kovarova M, Koller BH. Impaired host defense in mice lacking ONZIN. J Immunol 2007; 178(8): 5132-5143. https://doi.org/10.4049/jimmunol.178.8.5132
  23. Lee JH, Rho JI, Devkota S, Sung YH, Lee HW. Developing genetically engineered mouse models using engineered nucleases: Current status, challenges, and the way forward. Drug Discov Today: Disease Models 2016; 20: 13-20.
  24. Li C, Ma H, Wang Y, Cao Z, Graves-Deal R, Powell AE, Starchenko A, Ayers GD, Washington MK, Kamath V, Desai K, Gerdes MJ, Solnica-Krezel L, Coffey RJ. Excess PLAC8 promotes an unconventional ERK2-dependent EMT in colon cancer. J Clin Invest 2014; 124(5): 2172-2187. https://doi.org/10.1172/JCI71103
  25. McMurray HR, Sampson ER, Compitello G, Kinsey C, Newman L, Smith B, Chen SR, Klebanov L, Salzman P, Yakovlev A, Land H. Synergistic response to oncogenic mutations defines gene class critical to cancer phenotype. Nature 2008; 453(7198): 1112-1116. https://doi.org/10.1038/nature06973
  26. Michl P, Gress TM. Current concepts and novel targets in advanced pancreatic cancer. Gut 2013; 62(2): 317-326. https://doi.org/10.1136/gutjnl-2012-303588
  27. Miller KD, Siegel RL, Lin CC, Mariotto AB, Kramer JL, Rowland JH, Stein KD, Alteri R, Jemal A. Cancer treatment and survivorship statistics, 2016. CA Cancer J Clin 2016; 66(4): 271-289. https://doi.org/10.3322/caac.21349
  28. Muller U. Ten years of gene targeting: targeted mouse mutants, from vector design to phenotype analysis. Mech Dev 1999; 82(1-2): 3-21. https://doi.org/10.1016/S0925-4773(99)00021-0
  29. Paquet D, Kwart D, Chen A, Sproul A, Jacob S, Teo S, Olsen KM, Gregg A, Noggle S, Tessier-Lavigne M. Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9. Nature 2016; 533(7601): 125-129. https://doi.org/10.1038/nature17664
  30. Roh JI, Cheong C, Sung YH, Lee J, Oh J, Lee BS, Lee JE, Gho YS, Kim DK, Park CB, Lee JH, Lee JW, Kang SM, Lee HW. Perturbation of NCOA6 leads to dilated cardiomyopathy. Cell Rep 2014; 8(4): 991-998. https://doi.org/10.1016/j.celrep.2014.07.027
  31. Roh JI, Kim Y, Oh J, Kim Y, Lee J, Lee J, Chun KH, Lee HW. Hexokinase 2 is a molecular bridge linking telomerase and autophagy. PLoS One 2018; 13(2): e0193182. https://doi.org/10.1371/journal.pone.0193182
  32. Roh JI, Lee J, Park SU, Kang YS, Lee J, Oh AR, Choi DJ, Cha JY, Lee HW. CRISPR-Cas9-mediated generation of obese and diabetic mouse models. Exp Anim 2018; 67(2): 229-237. https://doi.org/10.1538/expanim.17-0123
  33. Segal AW. How neutrophils kill microbes. Annu Rev Immunol 2005; 23: 197-223. https://doi.org/10.1146/annurev.immunol.23.021704.115653
  34. Sung YH, Kim JM, Kim HT, Lee J, Jeon J, Jin Y, Choi JH, Ban YH, Ha SJ, Kim CH, Lee HW, Kim JS. Highly efficient gene knockout in mice and zebrafish with RNA-guided endonucleases. Genome Res 2014; 24(1): 125-131. https://doi.org/10.1101/gr.163394.113
  35. Valera A, Perales JC, Hatzoglou M, Bosch F. Expression of the neomycin-resistance (neo) gene induces alterations in gene expression and metabolism. Hum Gene Ther 1994; 5(4): 449-456. https://doi.org/10.1089/hum.1994.5.4-449
  36. Witko-Sarsat V, Rieu P, Descamps-Latscha B, Lesavre P, Halbwachs-Mecarelli L. Neutrophils: molecules, functions and pathophysiological aspects. Lab Invest 2000; 80(5): 617-653. https://doi.org/10.1038/labinvest.3780067
  37. Wright C, Simone NL. Obesity and tumor growth: inflammation, immunity, and the role of a ketogenic diet. Curr Opin Clin Nutr Metab Care 2016; 19(4): 294-299. https://doi.org/10.1097/MCO.0000000000000286
  38. Wu X, Kriz AJ, Sharp PA. Target specificity of the CRISPR-Cas9 system. Quant Biol 2014; 2(2): 59-70. https://doi.org/10.1007/s40484-014-0030-x
  39. Yoshiki A, Moriwaki K. Mouse phenome research: implications of genetic background. ILAR J 2006; 47(2): 94-102. https://doi.org/10.1093/ilar.47.2.94

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