Rapid growth cost in “all-fish” growth hormone gene transgenic carp: Reduced critical swimming speed

Evidence has accumulated that there is a trade-off between benefits and costs associated with rapid growth. A trade-off between growth rates and critical. swimming speed (U-crit) had been also reported to be common in teleost fish. We hypothesize that growth acceleration in the F-3 generation of "all-fish" growth hormone gene (GH) transgenic common carp (Cyprinus carpio L.) would reduce the swimming abilities. Growth and swimming performance between transgenic fish and non-transgenic controls were) compared. The results showed that transgenic fish had a mean body weight 1.4-1.9-fold heavier, and a mean specific growth rate (SGR) value 6%-10% higher than the controls. Transgenic fish, however, had a mean absolute U-crit (cm/s) value 22% or mean relative Ucrit (BL/s) value 24% lower than the controls. It suggested that fast-growing "all-fish" GH-transgenic carp were inferior swimmers. It is also supported that there was a trade-off between growth rates and swimming performance, i.e. faster-growing individuals had lower critical swimming speed.

Genetic analysis of all-fish growth hormone gene transferred carp (Cyprinus carpio L.) and its F-1 generation

Recombinant "all-fish" growth hormone gene (GH) was microinjected Into the fertilized eggs of carp. A comparison between the growth traits of transgenics and non-transgenics was carried out, and the transgenic individuals with significant "fast-growing" effect were successfully gained. A comparison on the reproductivities was also given out between the transgenics and their non-transgenic siblings, and showed that the reproductive capacity of transgenics was substantially equivalent to those of the non-transgenics. On the other hand, the genetic separation and the characteristic distribution of the F-1 generation were genetically analyzed, which gave solid evidence for the hypothesis that 2-3 chromosomes are integrated with transgene. In addition, the distinct biological effects for multisite-integrated transgenes were further discussed. The present study opens a door for the breeding of "fast-growing" transgenic fish.

Molecular evolution of GH in primates: characterisation of the GH genes from slow loris and marmoset defines an episode of rapid evolutionary change

Pituitary growth hormone (GH), like several other protein hormones, shows an unusual episodic pattern of molecular evolution in which sustained bursts of rapid change are imposed on long periods of very slow evolution (near-stasis). A marked period of rap

Growth and energy budget of F-2 'all-fish' growth hormone gene transgenic common carp

The growth and energy budget for F-2 'all-fish' growth hormone gene transgenic common carp Cyprinus carpio of two body sizes were investigated at 29.2 degrees C for 21 days. Specific growth rate, feed intake, feed efficiency, digestibility coefficients of dry matter and protein, gross energy intake (I-E), and the proportion of I-E utilized for heat production (H-E) were significantly higher in the transgenics than in the controls. The proportion of I-E directed to waste products [faecal energy (F-E) and excretory energy loss (Z(E) + U-E) where Z(E) is through the gills and U-E through the kidney], and the proportion of metabolizable energy (M-E) for recovered energy (R-E) were significantly lower in the transgenics than in the controls. The average energy budget equation of transgenic fish was as follows: 100 I-E = 19.3 F-E + 6.0 (Z(E) + U-E) + 45.2 H-E + 29.5 R-E or 100 M-E = 60.5 H-E + 39.5 R-E. The average energy budget equation of the controls was: 100 I-E = 25.2 F-E + 7.4 (Z(E) + U-E) + 35.5 H-E + 31.9 R-E or 100 M-E = 52.7 H-E + 47.3 R-E. These findings indicate that the high growth rate of 'all-fish' transgenic common carp relative to their non-transgenic counterparts was due to their increased feed intake, reduced lose of waste productions and improved feed efficiency. The benefit of the increased energy intake by transgenic fish, however, was diminished by their increased metabolism.

Molecular cloning of growth hormone receptor (GHR) from common carp (Cyprinus carpio L.) and identification of its two forms of mRNA transcripts

The cDNA of growth hormone receptor (GHR) was cloned from the liver of 2-year common carp (Cyprinus carpio L.) by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE). Its open reading frame (ORF) of 1806 nucleotides is translated into a putative peptide of 602 amino acids, including an extracellular ligand-binding domain of 244 amino acids (aa), a single transmembrane domain of 24 aa and an intracellular signal-transduction domain of 334 aa. Sequence analysis indicated that common carp GHR is highly homologous to goldfish (Carassius auratus) GHR at both gene and protein levels. Using a pair of gene-specific primers, a GHR fragment was amplified from the cDNA of 2-year common carp, a 224 bp product was identified in liver and a 321 bp product in other tissues. The sequencing of the products and the partial genomic DNA indicated that the difference in product size was the result of a 97 bp intron that alternatively spliced. In addition, the 321 bp fragment could be amplified from all the tissues of 4-month common carp including liver, demonstrating the occurrence of the alternative splicing of this intron during the development of common carp. Moreover, a semi-quantitative RT-PCR was performed to analyze the expression level of GHR in tissues of 2-year common carp and 4-month common carp. The result revealed that in the tissues of gill, thymus and brain, the expression level of GHR in 2-year common carp was significantly tower than that of 4-month common carp.

Effects of the "all-fish" growth hormone transgene expression on non-specific immune functions of common carp, Cyprinus carpio L.

This study investigated non-specific immune functions of the F-2 generation of "all-fish" growth hormone transgenic carp, Cyprinus carpio L. Lysozyme activity was 145.0 (+/- 30.7) U ml(-1) in the transgenic fish serum and 105.0 (+/- 38.7) U ml(-1) in age-matched non-transgenic control fish serum, a significant difference (P < 0.01). The serum bactericidal activity in the transgenics was significantly higher than that in the controls (P < 0.05), with the percentage serum killing of 59.5% (6.83%) and 50.8% (8.67%), respectively. Values for leukocrit and phagocytic percent of macrophages in head kidney were higher in transgenics than controls (P < 0.05). However, the phagocytic indices in the transgenics and the controls were not different. In addition, the mean body weight of the transgenics was 63.4 (6.65) g, much higher than that of the controls [39.2 (+/- 3.30) g, P < 0.01]. The absolute weight of spleen of the transgenics [0.13 (+/- 0.03) g] was higher than that of the controls [0.08 (+/- 0.02) g, P < 0.01]. However, there was no difference in the relative weight of spleen between the transgenics and the controls, with the spleen mass index being 0.21% (+/- 0.02%) and 0.20% (+/- 0.03%), respectively. This study suggests that the "all-fish" growth hormone transgene expression could stimulate not only the growth but also the non-specific immune functions of carp. (c) 2006 Published by Elsevier B.V.

Growth hormone gene transfer in common carp

The first successful case of transgenic fish was achieved in 1984. It is in a model system that the integration and expression of recombinant human growth hormone (hGH) in host red common carp (Cyprinus carpio, red var.) have been thoroughly studied. Recently, the integration sites have been recovered and characterized. Compared with non-transgenic peers, hGH-transgenic fish are prior in dietary utilization and growth performance. In view of bio-safety and bio-ethics, an "all-fish" construct CAgcGH, grass carp growth hormone fused with common carp P-actin promoter, has been generated and transferred into Yellow River carp (C carpio, local strain in Yellow River) fertilized eggs. Under middle-scale trial, CAgcGH-transgenics show higher growth rate and food conversion efficiency than the controls, which is consistent to laboratory findings. To avoid the potential impact of transgenic fish on the environment, a sterile strain of transgenic triploid fish has been successfully produced. The "all-fish" transgenic common carp is also approved safe enough as daily food, according to a test based on the pathological principles of new medicines issued by the Ministry of Health of China. The "all-fish" transgenic common carp with growth enhancement is now ready for market, but looking for governmental authorization. (C) 2003 Editions scientifiques et medicales Elsevier SAS and Ifremer/IRD/Inra/Cemagref. All rights reserved.

Whole-body amino acid pattern of F-4 human growth hormone gene-transgenic red common carp (Cyprinus carpio) fed diets with different protein levels

F-4 generation of human growth hormone (hGH) gene-transgenic red common carp, and the non-transgenic controls were fed for 8 weeks on purified diets with 20%, 30% or 40% protein. Analysis of whole-body amino acids showed that the proportions of lysine, leucine, phenylalanine, valine and alanine, as percentages of body protein, increased significantly, while those of arginine, glutamic acid and tyrosine decreased, with increases in dietary protein level in at least one strain of fish. Proportions of the other amino acids were unaffected by the diets. The proportions of lysine and arginine were significantly higher, while those of leucine and alanine were lower in the transgenics than in the controls in at least one diet group. Proportions of the other amino acids were unaffected by strain. The results suggest that the whole-body amino acid profile of transgenic carp, when expressed as proportions of body protein, was in general, similar to that of the non-transgenic controls. (C) 2000 Elsevier Science B.V. All rights reserved.

Growth and feed utilization by F-4 human growth hormone transgenic carp fed diets with different protein levels

The F-4 generation of human growth hormone (hGH) transgenic red common carp Cyprinus carpio had significantly higher growth rates than the non-transgenic controls. Protein and energy intakes were significantly higher in the transgenic carp than in the controls fed the 20% protein diet, but were not different between the two strains fed diets with 30 and 40% protein. Faecal protein loss, as a proportion of protein intake, was significantly lower in the transgenics than in the controls fed diets with 20 and 30% protein, but was not different between the two strains Fed diet with 40% protein. Faecal energy loss, as a proportion of energy intake, was significantly lower in the transgenics than in the controls fed diet with 20% protein, but was not different between the two strains fed diets with 30 and 40% protein. Recovered protein, as a proportion of protein intake, was significantly higher in the transgenics than in the controls fed all diets, whereas recovered energy was significantly higher in the transgenic fish fed the 40% protein diet. For fish fed each diet, the transgenics had significantly higher body contents of dry matter and protein, but lower contents of lipid than the controls. It was concluded that transgenics were more efficient in utilizing dietary protein than the controls. it a lower dietary protein level; transgenics achieved higher growth rates mainly by increasing feed intake; at higher levels of dietary protein, transgenics achieved higher growth rates mainly through a higher energy conversion efficiency. (C) 1998 The Fisheries Society of the British Isles.

HORMONAL REPLACEMENT THERAPY IN FISH - HUMAN GROWTH-HORMONE GENE-FUNCTION IN HYPOPHYSECTOMIZED CARP

Transgenic common carp, Cyprinus carpio, produced by the microinjection of fertilized eggs with a linearized chimeric plasmid pMThGH, a human growth hormone (hGH) gene with a mouse metallothionein-I (MT) gene promoter in pBR322, were used to produce F1 and F2 transgenics. Following hypophysectomy of the transgenic F2 common carp, non-transgenic common carp and non-transgenic crucian carp, growth was monitored for up to 110 days. In addition, recombinant hGH was injected subcutaenously into a group of the non-transgenic crucian carp. Growth rate analyses indicated that (1) hypophysectomy of non-transgenic common carp and crucian carp results in the cessation of growth, (2) hGH administration can stimulate the growth of hypophysectomized crucian carp and (3) hypophysectomized hGH-transgenic common carp continue to grow in the absence of their own growth hormone, suggesting that the hGH-transgene is being expressed in tissues other than the pituitary.

两种不同终止子在转基因鲤鱼中的促生长效应

转基因构建体中启动子的选择会直接影响转植基因的活性,近年来有研究表明转基因构建体中终止子的选择会一定程度地影响转植基因的活性。为了更好地筛选转基因构建体和培育快速生长的转"全鱼"生长激素(Growth hormone,GH)基因鱼,文章用鲤鱼β-actin基因终止子和生长激素基因终止子分别构建了转基因构建体,显微注射得到转"全鱼"GH基因鱼P0代养殖群体,比较两种不同终止子构建体的活性。统计分析发现,生长激素基因终止子构建体的养殖群体的体重频率呈正态分布且平均体重显著高于β-actin基因终止子构建体的养

Identification of the Angel-related elements in cyprinid fishes and their phylogenetic implications

Angel-related element belongs to the family of miniature inverted-repeat transposable elements (MITEs). In this paper we report the identification of an Angel-related element in the series Leuciscini of cyprinid fishes, which is located in the second intron of the growth hormone (GH) gene. We have also found that this element is absent in orthologous locus in the series Barbini of cyprinid fishes, that provides new evidence for the monophyly of the series Leuciscini. The insertion of Angel-related element into the GH gene might take place in the common ancestor of the series Leuciscini after its divergence from the series Barbini. The high sequence divergence and relatively broad species distribution of Angel-related elements implies that they might be ancient transposons which appeared about 26 million years ago.

Integration of double-fluorescence expression vectors into zebrafish genome for the selection of site-directed knockout/knockin

Production of zebrafish by modifying endogenous growth hormone (GH) gene through homologous recombination is described here. We first constructed the targeting vectors pGHT1.7k and pGHT2.8k, which were used for the knockout/knockin of the endogenous GH gene of zebrafish, and injected these two vectors into the embryos of zebrafish. Overall, the rate of targeted integration with the characteristic of germ line transmission in zebrafish was 1.7x10(-6). In one experimental patch, the integrating efficiency of pGHT2.8k was higher than that of pGHT1.7k, but the lethal effect of pGHT2.8k was stronger than that of pGHT1.7k. The clones with the correct integration of target genes were identified by a simple screening procedure based on green fluorescent protein (GFP) and RFP dual selection, which corresponded to homologous recombination and random insertion, respectively. The potential homologous recombination zebrafish was further bred to produce a heterozygous F-1 generation, selected based on the presence of GFP. The potential targeted integration of exogenous GH genes into a zebrafish genome at the P-0 generation was further verified by polymerase chain reaction and Southern blot analysis. Approximately 2.5% of potential founder knockout and knockin zebrafish had the characteristic of germ line transmission. In this study, we developed an efficient method for producing the targeted gene modification in zebrafish for future studies on genetic modifications and gene functions using this model organism.

Fast-growing transgenic common carp mounfing compensatory growth

Compensatory growth is a phase of accelerated growth apparent when favourable conditions are restored after a period of growth depression. To investigate if F-2 common 'all-fish' growth hormone gene transgenic common carp (Cyprinus carpio) could mount compensatory growth, a 9 week study at 29 degrees C was performed. The control group was fed to satiation twice a day throughout the experiment. The other two groups were deprived of feed for 1 or 2 weeks, respectively, and then fed to satiation during the re-feeding period. At the end of the experiment, the live masses of fish in the deprived groups were still significantly lower than those of the controls. During the re-feeding period, size-adjusted mean specific growth rates and mean feed intakes were significantly higher in the deprived fish than in the controls, indicating a partial compensatory growth response in these fish. No significant differences were found in food conversion efficiency between the deprived and control fish during re-feeding, suggesting that hyperphagia was the mechanism responsible for increased growth rates. The proximate composition of the deprived fish at the end of the experiment was similar to that of the control fish. This study is, to our knowledge, the first to report that fast-growing transgenic fish can achieve partial compensation of growth following starvation. (c) 2007 The Authors Journal compilation (c) 2007 The Fisheries Society of the British Isles.

哺乳动物生长激素/泌乳刺激素基因超家族的分子进化研究

生长激素（Growthhormone，GH）和泌乳刺激素（Prolactin，PRL）基因具有相似的结构和功能，它们和其他一些相关基因组成了GH/PRL基因超家族。在大部分哺乳动物基因组中，GH和PRL都是单拷贝基因。但在灵长目动物中，PRL是单拷贝基因，GH基因却发生了串联重复事件，形成一个基因家族。而在啮齿目中则相反，GH是单拷贝的，PRL却发生了串联重复事件形成一个基因家族。用PCR、克隆、测序的方法，我们从7种灵长目动物中得到了35条GH－类似基因。系统发育分析的结果显示所有旧大陆猴／人猿超科（OWM/H）的GH一类似基因和所有新大陆猴（NWM）的GH-类似基因分别形成两个单系，提示新大陆猴的GH基因家族和旧大陆猴／人猿超科GH基因家族起源于独立的基因重复事件。我们的分析结果还提示在。H基因家族的进化历程中发生了多次基因重复和基因转换事件。此外，不同GH基因家族成员的进化速率和所受到的选择压力存在显著差异。GHN基因在人猿超科和旧大陆猴中进化速率都比较慢，且受到了很强的纯化选择的作用；而CSH基因在两个世系中进化速率都比较快而且可能受到近中性选择的作用；GHV的进化速率和选择压力在旧大陆猴和人猿超科之间存在显著差异。对于新大陆猴GH基因家族，我们发现3个主要的功能基因簇，有趣的是这3个基因簇分别受到了纯化选择、近中性选择和正选择3种不同类型的选择压力。进一步分析的结果显示GH基因家族的进化符合所谓"生一和一灭（Birth-and-death）"的进化模式，该模式以频繁的基因重复和假基因化为主要特征。啮齿目泌乳刺激素基因家族由多个结构相似、在染色体上串联排列的基因组成，创门主要在生殖过程中协调作用。我们利用生物信息学手段在大鼠中得到了两个新的家族成员。结合系统发育、基因转换分析及染色体相对位置的比较，我们认为啮齿目PRL基因家族中的PL-I和PL-II基因亚家族是在大、小鼠分歧之后由物种特异的基因重复事件形成的。此外，啮齿目PRL基因家族的进化历程较复杂，因为除了通常的5-外显子结构的基因外，该家族还包含6-外显子结构的基因，后者在前者的第2和第3外显子之间获得了一个额外的外显子。本研究中我们意外地发现这个外显子在两个基因簇中的起源方式并不相同。在groupA中，它来源于一段外源DNA的插入，而在groupB中则来源于原先的非编码序列。对同义替换和异义替换速率比较的结果显示，在这些获得额外外显子的基－因中纯化选择压力得到了放松。激素蛋白必须与其受体结合经过信号传导才能发挥其生物学功能，因此，研究激素和受体的协同进化就显得尤为重要。我们对哺乳动物泌乳刺激素基因和其受体（PRLR）基因进行了协同进化分析，结果发现哺乳动物PRLR的膜外区和膜内区显示出和PRL一致的"插曲"式的进化模式。皮耳森相关系数计算的结果说明PRLR的膜外区和PRL基因发生了协同进化，同时PRLR的两个功能区域：膜外区和膜内区之间也发生了协同进化。此外，我们还发现灵长目PRL基因也发生了和GH基因类似的"插曲"式的进化，而且快速进化可能是选择压力放松的结果。