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.
英文摘要:
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.
1.Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, Wuhan 430072, Peoples R China 2.Univ Chinese Acad Sci, Beijing 100049, Peoples R China
