title: Targeted expression in zebrafish primordial germ cells by Cre/loxP and Gal4/UAS systems.
abstract: In zebrafish and other vertebrates, primordial germ cells (PGCs) are a population of embryonic cells that give rise to sperm and eggs in adults. Any type of genetically manipulated lines have to be originated from the germ cells of the manipulated founders, thus it is of great importance to establish an effective technology for highly specific PGC-targeted gene manipulation in vertebrates. In the present study, we used the Cre/loxP recombinase system and Gal4/UAS transcription system for induction and regulation of mRFP (monomer red fluorescent protein) gene expression to achieve highly efficient PGC-targeted gene expression in zebrafish. First, we established two transgenic activator lines, Tg(kop:cre) and Tg(kop:KalTA4), to express the Cre recombinases and the Gal4 activator proteins in PGCs. Second, we generated two transgenic effector lines, Tg(kop:loxP-SV40-loxP-mRFP) and Tg(UAS:mRFP), which intrinsically showed transcriptional silence of mRFP. When Tg(kop:cre) females were crossed with Tg(kop:loxP-SV40-loxP-mRFP) males, the loxP flanked SV40 transcriptional stop sequence was 100 % removed from the germ cells of the transgenic hybrids. This led to massive production of PGC-specific mRFP transgenic line, Tg(kop:loxP-mRFP), from an mRFP silent transgenic line, Tg(kop:loxP-SV40-loxP-mRFP). When Tg(kop:KalTA4) females were crossed with Tg(UAS:mRFP) males, the hybrid embryos showed PGC specifically expressed mRFP from shield stage till 25 days post-fertilization (pf), indicating the high sensitivity, high efficiency, and long-lasting effect of the Gal4/UAS system. Real-time PCR analysis showed that the transcriptional amplification efficiency of the Gal4/UAS system in PGCs can be about 300 times higher than in 1-day-pf embryos. More importantly, when the UAS:mRFP-nos1 construct was directly injected into the Tg(kop:KalTA4) embryos, it was possible to specifically label the PGCs with high sensitivity, efficiency, and persistence. Therefore, we have established two targeted gene expression platforms in zebrafish PGCs, which allows us to further manipulate the PGCs of zebrafish at different levels.
authors.affiliation: State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, China.
authors.forename: Feng
authors.initials: F
authors.lastname: Xiong
authors.forename: Zhi-Qiang
authors.initials: ZQ
authors.lastname: Wei
authors.forename: Zuo-Yan
authors.initials: ZY
authors.lastname: Zhu
authors.forename: Yong-Hua
authors.initials: YH
authors.lastname: Sun
mesh_terms: D000818:Animals; D003433:Crosses, Genetic; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005260:Female; D015870:Gene Expression; D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D005854:Germ Cells / Q000378:metabolism*; D019426:Integrases / Q000378:metabolism*; D008164:Luminescent Proteins; D008297:Male; D060888:Real-Time Polymerase Chain Reaction; D013194:Staining and Labeling; D014157:Transcription Factors / Q000378:metabolism*; D015027:Zebrafish / Q000235:genetics / Q000378:metabolism*; D029961:Zebrafish Proteins / Q000378:metabolism*; D000097573:Red Fluorescent Protein
chemical_list: D004268:DNA-Binding Proteins; D019426:Integrases; D008164:Luminescent Proteins; D014157:Transcription Factors; D029961:Zebrafish Proteins; D000097573:Red Fluorescent Protein; C542436:Gal4 protein, zebrafish; C045073:Cre recombinase
references.citation: Proc Natl Acad Sci U S A. 2005 Mar 15;102(11):4074-9
references.pmid: 15728735
references.citation: Genesis. 2007 Jun;45(6):413-7
references.pmid: 17551945
references.citation: Biol Reprod. 2008 Jan;78(1):159-66
references.pmid: 17901077
references.citation: FASEB J. 2010 Aug;24(8):2641-50
references.pmid: 20371629
references.citation: Transgenic Res. 2005 Apr;14(2):217-23
references.pmid: 16022392
references.citation: Curr Biol. 2002 Mar 19;12(6):454-66
references.pmid: 11909530
references.citation: Nature. 2005 May 19;435(7040):360-4
references.pmid: 15902260
references.citation: Proc Natl Acad Sci U S A. 1992 Jul 15;89(14):6232-6
references.pmid: 1631115
references.citation: Dev Growth Differ. 2005 Jun;47(5):323-31
references.pmid: 16026540
references.citation: Cancer Sci. 2010 Jan;101(1):149-54
references.pmid: 19961491
references.citation: Dev Dyn. 2005 Aug;233(4):1366-77
references.pmid: 15977183
references.citation: Cell. 2007 Dec 28;131(7):1273-86
references.pmid: 18155131
references.citation: J Biol Chem. 1997 Feb 28;272(9):5695-702
references.pmid: 9038180
references.citation: Proc Natl Acad Sci U S A. 2002 Nov 12;99(23):14919-24
references.pmid: 12397179
references.citation: Mech Dev. 2000 Jul;95(1-2):271-4
references.pmid: 10906476
references.citation: Genetics. 1996 Oct;144(2):715-26
references.pmid: 8889532
references.citation: Plant Biotechnol J. 2007 Mar;5(2):263-274
references.pmid: 17309681
references.citation: Mar Biotechnol (NY). 2010 Feb;12(1):1-7
references.pmid: 19590921
references.citation: Dev Dyn. 2007 Nov;236(11):3088-99
references.pmid: 17937395
references.citation: Curr Biol. 2003 Aug 19;13(16):1429-34
references.pmid: 12932328
references.citation: J Cell Sci. 2005 Sep 1;118(Pt 17):4027-38
references.pmid: 16129886
references.citation: Proc Natl Acad Sci U S A. 1982 Dec;79(23):7410-4
references.pmid: 6760197
references.citation: Dev Dyn. 2008 Aug;237(8):2139-46
references.pmid: 18627109
references.citation: Genetics. 2006 Oct;174(2):639-49
references.pmid: 16959904
references.citation: EMBO Rep. 2006 May;7(5):496-9
references.pmid: 16670683
references.citation: J Mol Biol. 1985 Feb 5;181(3):351-62
references.pmid: 3856690
references.citation: Development. 1997 Aug;124(16):3157-65
references.pmid: 9272956
references.citation: Nat Methods. 2007 Apr;4(4):323-6
references.pmid: 17369834
references.citation: Proc Natl Acad Sci U S A. 2005 Apr 26;102(17):6068-73
references.pmid: 15827121
references.citation: Cell. 1983 Apr;32(4):1301-11
references.pmid: 6220808
references.citation: Mar Biotechnol (NY). 2007 Jul-Aug;9(4):420-8
references.pmid: 17503154
references.citation: Development. 2011 Nov;138(22):4831-41
references.pmid: 22007133
references.citation: Methods. 1998 Apr;14(4):367-79
references.pmid: 9608508
references.citation: Stem Cells Dev. 2008 Jun;17(3):585-97
references.pmid: 18576915
references.citation: Dev Biol. 2007 Apr 15;304(2):811-24
references.pmid: 17335798
references.citation: Dev Dyn. 1995 Jul;203(3):253-310
references.pmid: 8589427
references.citation: Cell. 2007 Dec 28;131(7):1226-7
references.pmid: 18160032
references.citation: Methods Cell Biol. 2004;77:505-19
references.pmid: 15602929
references.citation: J Mol Biol. 1989 Oct 5;209(3):423-32
references.pmid: 2511324
references.citation: Dev Cell. 2004 Jan;6(1):7-28
references.pmid: 14723844
references.citation: Gene. 1989 Apr 15;77(1):61-8
references.pmid: 2744488
references.citation: Dev Dyn. 2008 Sep;237(9):2460-5
references.pmid: 18729206
references.citation: Cell. 1985 Apr;40(4):767-74
references.pmid: 3886158
references.citation: Nucleic Acids Res. 1998 Jun 1;26(11):2729-34
references.pmid: 9592161
references.citation: Methods. 2001 Dec;25(4):402-8
references.pmid: 11846609
references.citation: Proc Natl Acad Sci U S A. 2007 May 29;104(22):9410-5
references.pmid: 17517602
references.citation: Mech Dev. 2002 Aug;116(1-2):141-50
references.pmid: 12128213
references.citation: Proc Natl Acad Sci U S A. 2008 Jan 29;105(4):1255-60
references.pmid: 18202183
references.citation: Genesis. 2009 Sep;47(9):617-27
references.pmid: 19548313
references.citation: Curr Biol. 1998 Feb 12;8(4):243-6
references.pmid: 9501989
references.citation: Proc Natl Acad Sci U S A. 2009 Aug 11;106(32):13365-70
references.pmid: 19628697
references.citation: Science. 1994 Jul 1;265(5168):103-6
references.pmid: 8016642
references.citation: Nature. 2005 Jul 14;436(7048):207-13
references.pmid: 15937476
references.citation: Genes Dev. 2001 Nov 1;15(21):2877-85
references.pmid: 11691838
references.citation: Dev Biol. 2008 Jun 1;318(1):133-42
references.pmid: 18436203
references.citation: Dev Biol. 2008 Sep 1;321(1):197-204
references.pmid: 18601916
references.citation: Dev Dyn. 2008 Oct;237(10):2955-62
references.pmid: 18816824
references.citation: Genesis. 2000 Feb;26(2):116-7
references.pmid: 10686602