The genetic stability and expression efficiency of exogenous genes in transgenic animals are closely related to integration site and copy number. In our laboratory, by transgenic manipulation and subsequent test cross...The genetic stability and expression efficiency of exogenous genes in transgenic animals are closely related to integration site and copy number. In our laboratory, by transgenic manipulation and subsequent test crosses, we established an ‘‘all-fish'' growth hormone(GH)transgenic common carp family that exhibits fast growth.In this present study, genome walking, real-time quantitative polymerase chain reaction, and fluorescence in situ hybridization techniques were applied to identify the integration characteristics of the exogenous grass carp GH gene in the transgenic common carp. The exogenous GH genes, in the form of two complete and one incomplete tandem repeats, were found to have integrated into an ATrich region near the end of a chromosome pair. We hypothesize that the high efficiency of exogenous GH gene expression might be due to the low copy number in the genome and the AT-rich integration site.展开更多
Ammonia is toxic to fish in natural and artificial waters.We evaluated the acute(96 h) and chronic(21 d) toxicity of un-ionized ammonia to GH transgenic common carp(Cyprinus carpio L.) and non-transgenic common carp u...Ammonia is toxic to fish in natural and artificial waters.We evaluated the acute(96 h) and chronic(21 d) toxicity of un-ionized ammonia to GH transgenic common carp(Cyprinus carpio L.) and non-transgenic common carp using a static-renewal bioassay.The 24,48,72 and 96 h median lethal concentrations(LC50) of un-ionized ammonia were slightly lower in transgenic carp(2.64,2.44,2.28 and 2.16 mg N/L,respectively) than in non-transgenic carp(2.70,2.64,2.52 and 2.33 mg N/L,respectively).Similarly,the median lethal time(LT50) was significantly shorter for transgenic carp(1.41,7.91 and 117.42 h) than for non-transgenic common carp(2.53,14.06 and 150.44 h) following exposure to 3.86,3.29,or 2.09 mg N/L,respectively.Moreover,the mortality of transgenic carp was significantly higher than that of non-transgenic carp at all un-ionized ammonia concentrations((0.91 ± 0.12),(0.48 ± 0.06) and(0.12 ± 0.01) mg N/L) during the 21 d chronic toxicity test.Our results suggest that GH transgenic carp are less tolerant of unionized ammonia than non-transgenic carp.Our data are useful for evaluating potential environmental risk,optimizing stocking density in intensive aquaculture and establishing water quality criteria for ammonia in aquaculture.展开更多
The aim of the present study was to purify the common native carp growth hormone (ncGH), produce monoclonal antibodies (mAbs) to common native carp growth hormone (ncGH), and further enhance the sensitivity of enzyme-...The aim of the present study was to purify the common native carp growth hormone (ncGH), produce monoclonal antibodies (mAbs) to common native carp growth hormone (ncGH), and further enhance the sensitivity of enzyme-linked immunosorbent assays (ELISA) for ncGH. Additionally, we investigated changes in serum ncGH levels in carps raised in different environmental conditions. The recombinant grass carp (Ctenopharyngodon idella) growth hormone was purified and used as antigen to immunize the rabbit. The natural ncGH was isolated from the pituitaries of common carp. SDS-PAGE and Western blot utilizing the polyclonal anti-rgcGH antibody confirmed the purification of ncGH from pituitaries. Purified ncGH was then used as an immunogen in the B lymphocyte hybridoma technique. A total of 14 hybridoma cell lines (FMU-cGH 1-14) were established that were able to stably secrete mAbs against ncGH. Among them, eight clones (FMU-cGH1-6, 12 and 13) were successfully used for Western blot while nine clones (FMU-cGH 1-7, 9 and 10) were used in fluorescent staining and immunohistochemistry. Epitope mapping by competitive ELISA demonstrated that these mAbs recognized five different epitopes. A sensitive sandwich ELISA for detection of ncGH was developed using FMU-cGH12 as the coating mAb and FMU-cGH6 as the enzyme labeled mAb. This detection system was found to be highly stable and sensitive, with detection levels of 70 pg/mL. Additionally, we found that serum ncGH levels in restricted food group and in the net cage group increased 6.9-and 5.8-fold, respectively, when compared to controls, demonstrating differences in the GH stress response in common carp under different living conditions.展开更多
Gonadotropin-releasing hormone (GnRH) is a conservative neurodecapeptide fam-ily, which plays a crucial role in regulating the gonad development and in controlling the final sexual maturation in vertebrate. Two differ...Gonadotropin-releasing hormone (GnRH) is a conservative neurodecapeptide fam-ily, which plays a crucial role in regulating the gonad development and in controlling the final sexual maturation in vertebrate. Two differing cGnRH-II cDNAs of common carp, namely cGnRH-II cDNA1 and cDNA2, were firstly cloned from the brain by rapid amplification of cDNA end (RACE) and reverse transcription- polymerase chain reaction (RT-PCR). The length of cGnRH-II cDNA1 and cDNA2 was 622 and 578 base pairs (bp), respectively. The cGnRH-II pre-cursors encoded by two cDNAs consisted of 86 amino acids, including a signal peptide, cGnRH-II decapeptide and a GnRH-associated peptide (GAP) linked by a Gly-Lys-Arg proteolytic site. The results of intron trapping and Southern blot showed that two differing cGnRH-II genes in common carp genome were further identified, and that two genes might exist as a single copy. The multi-gene coding of common carp cGnRH-II gene offered novel evidence for gene duplica-tion hypothesis. Using semi-quantitative RT-PCR, expression and relative expression levels of cGnRH-II genes were detected in five dissected brain regions, pituitary and gonad of common carp. With the exception of no mRNA2 in ovary, two cGnRH-II genes could be expressed in all the detected tissues. However, expression levels showed an apparent difference in different brain regions, pituitary and gonad. According to the expression characterization of cGnRH-II genes in brain areas, it was presumed that cGnRH-II might mainly work as the neurotransmitter and neuromodulator and also operate in the regulation for the GnRH releasing. Then, the ex-pression of cGnRH-II genes in pituitary and gonad suggested that cGnRH-II might act as the autocrine or paracrine regulator.展开更多
基金supported by the National High Technology Research and Development Program of China(‘‘863’’Program)(2011AA100404,2011AA100403)the National Natural Science Foundation of China(31325026,31272649)
文摘The genetic stability and expression efficiency of exogenous genes in transgenic animals are closely related to integration site and copy number. In our laboratory, by transgenic manipulation and subsequent test crosses, we established an ‘‘all-fish'' growth hormone(GH)transgenic common carp family that exhibits fast growth.In this present study, genome walking, real-time quantitative polymerase chain reaction, and fluorescence in situ hybridization techniques were applied to identify the integration characteristics of the exogenous grass carp GH gene in the transgenic common carp. The exogenous GH genes, in the form of two complete and one incomplete tandem repeats, were found to have integrated into an ATrich region near the end of a chromosome pair. We hypothesize that the high efficiency of exogenous GH gene expression might be due to the low copy number in the genome and the AT-rich integration site.
基金supported by the National Basic Research Program of China (2007CB109205,2009CB118804)the National Natural Science Foundation of China (30623001)
文摘Ammonia is toxic to fish in natural and artificial waters.We evaluated the acute(96 h) and chronic(21 d) toxicity of un-ionized ammonia to GH transgenic common carp(Cyprinus carpio L.) and non-transgenic common carp using a static-renewal bioassay.The 24,48,72 and 96 h median lethal concentrations(LC50) of un-ionized ammonia were slightly lower in transgenic carp(2.64,2.44,2.28 and 2.16 mg N/L,respectively) than in non-transgenic carp(2.70,2.64,2.52 and 2.33 mg N/L,respectively).Similarly,the median lethal time(LT50) was significantly shorter for transgenic carp(1.41,7.91 and 117.42 h) than for non-transgenic common carp(2.53,14.06 and 150.44 h) following exposure to 3.86,3.29,or 2.09 mg N/L,respectively.Moreover,the mortality of transgenic carp was significantly higher than that of non-transgenic carp at all un-ionized ammonia concentrations((0.91 ± 0.12),(0.48 ± 0.06) and(0.12 ± 0.01) mg N/L) during the 21 d chronic toxicity test.Our results suggest that GH transgenic carp are less tolerant of unionized ammonia than non-transgenic carp.Our data are useful for evaluating potential environmental risk,optimizing stocking density in intensive aquaculture and establishing water quality criteria for ammonia in aquaculture.
基金the Development Plan of the State Key Fundamental Research ofChina(Grant No.2007CB109206)Chinese National Programs for High Technology Research and Development(Grant No.2006AA10Z141)the National NaturalScience Foundation of China(Grant No.30130050)
文摘The aim of the present study was to purify the common native carp growth hormone (ncGH), produce monoclonal antibodies (mAbs) to common native carp growth hormone (ncGH), and further enhance the sensitivity of enzyme-linked immunosorbent assays (ELISA) for ncGH. Additionally, we investigated changes in serum ncGH levels in carps raised in different environmental conditions. The recombinant grass carp (Ctenopharyngodon idella) growth hormone was purified and used as antigen to immunize the rabbit. The natural ncGH was isolated from the pituitaries of common carp. SDS-PAGE and Western blot utilizing the polyclonal anti-rgcGH antibody confirmed the purification of ncGH from pituitaries. Purified ncGH was then used as an immunogen in the B lymphocyte hybridoma technique. A total of 14 hybridoma cell lines (FMU-cGH 1-14) were established that were able to stably secrete mAbs against ncGH. Among them, eight clones (FMU-cGH1-6, 12 and 13) were successfully used for Western blot while nine clones (FMU-cGH 1-7, 9 and 10) were used in fluorescent staining and immunohistochemistry. Epitope mapping by competitive ELISA demonstrated that these mAbs recognized five different epitopes. A sensitive sandwich ELISA for detection of ncGH was developed using FMU-cGH12 as the coating mAb and FMU-cGH6 as the enzyme labeled mAb. This detection system was found to be highly stable and sensitive, with detection levels of 70 pg/mL. Additionally, we found that serum ncGH levels in restricted food group and in the net cage group increased 6.9-and 5.8-fold, respectively, when compared to controls, demonstrating differences in the GH stress response in common carp under different living conditions.
基金This work was supported by the'863'High Technology Project of China(Grant No.2001AA213101,2001AA212281)the National Natural Science Foundation of China(Grant No.200102006)+1 种基金the'973'Project of the Ministry of ScienceTechnology(Grant No.2001 CB 109006).
文摘Gonadotropin-releasing hormone (GnRH) is a conservative neurodecapeptide fam-ily, which plays a crucial role in regulating the gonad development and in controlling the final sexual maturation in vertebrate. Two differing cGnRH-II cDNAs of common carp, namely cGnRH-II cDNA1 and cDNA2, were firstly cloned from the brain by rapid amplification of cDNA end (RACE) and reverse transcription- polymerase chain reaction (RT-PCR). The length of cGnRH-II cDNA1 and cDNA2 was 622 and 578 base pairs (bp), respectively. The cGnRH-II pre-cursors encoded by two cDNAs consisted of 86 amino acids, including a signal peptide, cGnRH-II decapeptide and a GnRH-associated peptide (GAP) linked by a Gly-Lys-Arg proteolytic site. The results of intron trapping and Southern blot showed that two differing cGnRH-II genes in common carp genome were further identified, and that two genes might exist as a single copy. The multi-gene coding of common carp cGnRH-II gene offered novel evidence for gene duplica-tion hypothesis. Using semi-quantitative RT-PCR, expression and relative expression levels of cGnRH-II genes were detected in five dissected brain regions, pituitary and gonad of common carp. With the exception of no mRNA2 in ovary, two cGnRH-II genes could be expressed in all the detected tissues. However, expression levels showed an apparent difference in different brain regions, pituitary and gonad. According to the expression characterization of cGnRH-II genes in brain areas, it was presumed that cGnRH-II might mainly work as the neurotransmitter and neuromodulator and also operate in the regulation for the GnRH releasing. Then, the ex-pression of cGnRH-II genes in pituitary and gonad suggested that cGnRH-II might act as the autocrine or paracrine regulator.
