• ISSN 1674-8301
  • CN 32-1810/R
Volume 35 Issue 2
Mar.  2021
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Article Contents
Amy T. Desaulniers, Rebecca A. Cederberg, Elizabeth P. Carreiro, Channabasavaiah B. Gurumurthy, Brett R. White. A transgenic pig model expressing a CMV-ZsGreen1 reporter across an extensive array of tissues[J]. The Journal of Biomedical Research, 2021, 35(2): 163-173. doi: 10.7555/JBR.34.20200111
Citation: Amy T. Desaulniers, Rebecca A. Cederberg, Elizabeth P. Carreiro, Channabasavaiah B. Gurumurthy, Brett R. White. A transgenic pig model expressing a CMV-ZsGreen1 reporter across an extensive array of tissues[J]. The Journal of Biomedical Research, 2021, 35(2): 163-173. doi: 10.7555/JBR.34.20200111

A transgenic pig model expressing a CMV-ZsGreen1 reporter across an extensive array of tissues

doi: 10.7555/JBR.34.20200111
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  • Corresponding author: Brett R. White, Department of Animal Science, University of Nebraska-Lincoln, 3940 Fair Street, Lincoln, NE 68583-0908, USA. Tel: +1-402-472-6438, E-mail: bwhite2@unl.edu
  • Received: 2020-07-16
  • Revised: 2020-10-13
  • Accepted: 2020-11-10
  • Published: 2020-12-25
  • Issue Date: 2021-03-26
  • Since genetic engineering of pigs can benefit both biomedicine and agriculture, selecting a suitable gene promoter is critically important. The cytomegalovirus (CMV) promoter, which can robustly drive ubiquitous transgene expression, is commonly used at present, yet recent reports suggest tissue-specific activity in the pig. The objective of this study was to quantify ZsGreen1 protein (in lieu of CMV promoter activity) in tissues from pigs harboring a CMV-ZsGreen1 transgene with a single integration site. Tissue samples (n=35) were collected from neonatal hemizygous (n=3) and homozygous (n=3) piglets and ZsGreen1 abundance was determined via immunoblotting. ZsGreen1 was detected in all tissues, except hypothalamus, kidney cortex and oviduct. The expression patterns of homozygous and hemizygous piglets were similar (P>0.05). However, quantification revealed that ZsGreen1 protein levels were tissue-specific. Within neural/endocrine tissues, ZsGreen1 abundance was highest in the anterior pituitary gland, intermediate in the cerebellum and lowest in the cerebrum, spinal cord and posterior pituitary (P<0.05). In the digestive system, ZsGreen1 was more abundant in the salivary gland than esophagus, stomach, pancreas, duodenum, jejunum, ileum, spleen, colon, gallbladder and liver (P<0.05). Interestingly, ZsGreen1 amounts also differed within an organ (i.e., the right ventricle had 3-fold higher levels than the other heart chambers; P<0.05). These results provide useful information for the use of the CMV promoter to drive transgene expression in the pig. Moreover, this swine model represents a novel resource of ZsGreen1-labeled organs and a valuable tool to advance genome editing research.


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  • [1]
    Walters EM, Wells KD, Bryda EC, et al. Swine models, genomic tools and services to enhance our understanding of human health and diseases[J]. Lab Anim (NY), 2017, 46(4): 167–172. doi: 10.1038/laban.1215
    Desaulniers AT, Cederberg RA, Mills GA, et al. Production of a gonadotropin-releasing hormone 2 receptor knockdown (GNRHR2 KD) swine line[J]. Transgenic Res, 2017, 26(4): 567–575. doi: 10.1007/s11248-017-0023-4
    Bleck GT, White BR, Miller DJ, et al. Production of bovine α-lactalbumin in the milk of transgenic pigs[J]. J Anim Sci, 1998, 76(12): 3072–3078. doi: 10.2527/1998.76123072x
    Whitworth KM, Rowland RR, Ewen CL, et al. Gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus[J]. Nat Biotechnol, 2016, 34(1): 20–22. doi: 10.1038/nbt.3434
    Matz MV, Fradkov AF, Labas YA, et al. Fluorescent proteins from nonbioluminescent Anthozoa species[J]. Nat Biotechnol, 1999, 17(10): 969–973. doi: 10.1038/13657
    Day RN, Davidson MW. The fluorescent protein palette: tools for cellular imaging[J]. Chem Soc Rev, 2009, 38(10): 2887–2921. doi: 10.1039/b901966a
    Nakamura Y, Ishii J, Kondo A. Bright fluorescence monitoring system utilizing Zoanthus sp. green fluorescent protein (ZsGreen) for human G-protein-coupled receptor signaling in microbial yeast cells[J]. PLoS One, 2013, 8(12): e82237. doi: 10.1371/journal.pone.0082237
    Wouters M, Smans K, Vanderwinden JM. WZsGreen/+: a new green fluorescent protein knock-in mouse model for the study of KIT-expressing cells in gut and cerebellum[J]. Physiol Genomics, 2005, 22(3): 412–421. doi: 10.1152/physiolgenomics.00105.2005
    Boshart M, Weber F, Jahn G, et al. A very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus[J]. Cell, 1985, 41(2): 521–530. doi: 10.1016/S0092-8674(85)80025-8
    Brown AJ, Sweeney B, Mainwaring DO, et al. NF-κB, CRE and YY1 elements are key functional regulators of CMV promoter-driven transient gene expression in CHO cells[J]. Biotechnol J, 2015, 10(7): 1019–1028. doi: 10.1002/biot.201400744
    Mella-Alvarado V, Gautier A, Le Gac F, et al. Tissue and cell-specific transcriptional activity of the human cytomegalovirus immediate early gene promoter (UL123) in zebrafish[J]. Gene Expr Patterns, 2013, 13(3-4): 91–103. doi: 10.1016/j.gep.2013.01.003
    Liu CX, Wang LQ, Li WR, et al. Highly efficient generation of transgenic sheep by lentivirus accompanying the alteration of methylation status[J]. PLoS One, 2013, 8(1): e54614. doi: 10.1371/journal.pone.0054614
    Duan B, Cheng L, Gao Y, et al. Silencing of fat-1 transgene expression in sheep may result from hypermethylation of its driven cytomegalovirus (CMV) promoter[J]. Theriogenology, 2012, 78(4): 793–802. doi: 10.1016/j.theriogenology.2012.03.027
    Dyck MK, Ouellet M, Gagné M, et al. Testes-specific transgene expression in insulin-like growth factor-I transgenic mice[J]. Mol Reprod Dev, 1999, 54(1): 32–42. doi: 10.1002/(SICI)1098-2795(199909)54:1<32::AID-MRD5>3.0.CO;2-U
    Villuendas G, Gutiérrez-Adán A, Jiménez A, et al. CMV-driven expression of green fluorescent protein (GFP) in male germ cells of transgenic mice and its effect on fertility[J]. Int J Androl, 2001, 24(5): 300–305. doi: 10.1046/j.1365-2605.2001.00302.x
    Charreau B, Tesson L, Buscail J, et al. Analysis of human CD59 tissue expression directed by the CMV-IE-1 promoter in transgenic rats[J]. Transgenic Res, 1996, 5(6): 443–450. doi: 10.1007/BF01980209
    McGrew MJ, Sherman A, Ellard FM, et al. Efficient production of germline transgenic chickens using lentiviral vectors[J]. EMBO Rep, 2004, 5(7): 728–733. doi: 10.1038/sj.embor.7400171
    Vasey DB, Lillico SG, Sang HM, et al. CMV enhancer-promoter is preferentially active in exocrine cells in vivo[J]. Transgenic Res, 2009, 18(2): 309–314. doi: 10.1007/s11248-008-9235-y
    Whitelaw CBA, Radcliffe PA, Ritchie WA, et al. Efficient generation of transgenic pigs using equine infectious anaemia virus (EIAV) derived vector[J]. FEBS Lett, 2004, 571(1-3): 233–236. doi: 10.1016/j.febslet.2004.06.076
    Hsu WL, Johnson RK. Analysis of 28 generations of selection for reproduction, growth, and carcass traits in swine[J]. J Anim Sci, 2014, 92(11): 4806–4822. doi: 10.2527/jas.2014-8125
    Desaulniers AT, Cederberg RA, Mills GA, et al. LH-independent testosterone secretion is mediated by the interaction between GnRH2 and its receptor within porcine testes[J]. Biol Reprod, 2015, 93(2): 45. doi: 10.1095/biolreprod.115.128082
    Eaton SL, Roche SL, Llavero Hurtado M, et al. Total protein analysis as a reliable loading control for quantitative fluorescent Western blotting[J]. PLoS One, 2013, 8(8): e72457. doi: 10.1371/journal.pone.0072457
    Furth PA, Hennighausen L, Baker C, et al. The variability in activity of the universally expressed human cytomegalovirus immediate early gene 1 enhancer/promoter in transgenic mice[J]. Nucleic Acids Res, 1991, 19(22): 6205–6208. doi: 10.1093/nar/19.22.6205
    Dobie KW, Lee M, Fantes JA, et al. Variegated transgene expression in mouse mammary gland is determined by the transgene integration locus[J]. Proc Natl Acad Sci U S A, 1996, 93(13): 6659–6664. doi: 10.1073/pnas.93.13.6659
    Garrick D, Fiering S, Martin DI, et al. Repeat-induced gene silencing in mammals[J]. Nat Genet, 1998, 18(1): 56–59. doi: 10.1038/ng0198-56
    Chang SP, Opsahl ML, Whitelaw CB, et al. Relative transgene expression frequencies in homozygous versus hemizygous transgenic mice[J]. Transgenic Res, 2013, 22(6): 1143–1154. doi: 10.1007/s11248-013-9732-5
    Isern E, Gustems M, Messerle M, et al. The activator protein 1 binding motifs within the human cytomegalovirus major immediate-early enhancer are functionally redundant and act in a cooperative manner with the NF-κB sites during acute infection[J]. J Virol, 2011, 85(4): 1732–1746. doi: 10.1128/JVI.01713-10
    Rodova M, Jayini R, Singasani R, et al. CMV promoter is repressed by p53 and activated by JNK pathway[J]. Plasmid, 2013, 69(3): 223–230. doi: 10.1016/j.plasmid.2013.01.004
    Landolfo S, Gariglio M, Gribaudo G, et al. The human cytomegalovirus[J]. Pharmacol Ther, 2003, 98(3): 269–297. doi: 10.1016/S0163-7258(03)00034-2
    Pierzynowski SG, Weström BR, Erlanson-Albertsson C, et al. Induction of exocrine pancreas maturation at weaning in young developing pigs[J]. J Pediatr Gastroenterol Nutr, 1993, 16(3): 287–293. doi: 10.1097/00005176-199304000-00012
    Opsahl ML, Springbett A, Lathe R, et al. Mono-allelic expression of variegating transgene locus in the mouse[J]. Transgenic Res, 2003, 12(6): 661–669. doi: 10.1023/B:TRAG.0000005166.74030.ba
    Velten J, Cakir C, Youn E, et al. Transgene silencing and transgene-derived siRNA production in tobacco plants homozygous for an introduced AtMYB90 construct[J]. PLoS One, 2012, 7(2): e30141. doi: 10.1371/journal.pone.0030141
    Brodersen P, Voinnet O. The diversity of RNA silencing pathways in plants[J]. Trends Genet, 2006, 22(5): 268–280. doi: 10.1016/j.tig.2006.03.003
    Takahashi G, Gurumurthy CB, Wada K, et al. GONAD: genome-editing via Oviductal Nucleic Acids Delivery system: a novel microinjection independent genome engineering method in mice[J]. Sci Rep, 2015, 5: 11406. doi: 10.1038/srep11406
    Quadros RM, Miura H, Harms DW, et al. Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins[J]. Genome Biol, 2017, 18(1): 92. doi: 10.1186/s13059-017-1220-4
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