A perspective on fish gonad manipulation for biotechnical applications

The gonad is an essential organ for generating sperm and ova in vertebrates. This review describes several pilot studies on gonad gene manipulation and development in fish. With antisense RNA techniques, we suppressed the gonad development, and thus the fertility, of an antisense gonadotropin-releasing hormone (sGnRH) transgenic common carp. Then, using a tissue-specific exogenous gene excision strategy with sexual compensation, we knocked out the gonad-specific transgene. Under the control of the rainbow trout protamine promoter, the transgenic fish expressed the reporter gene eGFP specifically in the spermary. These results indicate that the fish gonad is a new model organ that can improve contemporary biotechnology experiments. Herein we discuss the potential of fish gonad manipulation for resolving important biosafety problems regarding transgenic fish generation and producing the new transgenic animal bioreactor.

Expression and molecular analysis of phbB gene in Chlamydomonas reinhardtii

The expression vector containing phbB and ble genes was constructed and transformed into cell-wall-deficient strain Chlamydomonas reinhardtii CC-849 by the glass-head method. The transgenic alga was selected and maintained in the TAP agar plates containing 10 mug/mL Zeomycin. Transgenic alga, which could express phbB at the transcriptional level, was obtained and further confirmed with PCR, Southern blot and RT-PCR-DNA hybridization analysis.

Cloning of rainbow trout (Oncorhynchus mykiss) histone H3 promoter and the activity analysis in rare minnow (Gobiocypris rarus)

Rainbow trout historic H3 (RH3) promoter was cloned via high fidelity PCR. The cloned RH3 promoter was inserted into a promoter-lacked vector pEGFP-1, resulting in an expression vector pRH3FGFP-1. The linearized pRH3EGFP-1 was microinjected into fertilized eggs of rare minnows and the sequential embryogenetic processes were monitored under a fluorescent microscope. Strong green fluorescence was ubiquitously observed at as early as the gastrula stage and then in various tissues at the fry stage. The results indicate that RH3 promoter, as a piscine promoter, could serve in producing transgenic Cyprinoid such as rare minnow. Promoter activity of RH3, CMV and common carp beta-actin (CA) were compared in rare minnow by the expression of respective recombinant EGFP vectors. The expression of pCMVEGFP occurred earlier than the following one, pRH3EGFP-1, and then pCAEGFP during the embryogenesis of the transgenics. Their expression activities demonstrated that the CMV promoter is the strongest one, followed by the CA and then the RH3.

Retention of the developmental pluripotency in medaka embryonic stem cells after gene transfer and long-term drug selection for gene targeting in fish

Embryonic stem (ES) cells provide a unique tool for introducing random or targeted genetic alterations, because it is possible that the desired, but extremely rare recombinant genotypes can be screened by drug selection. ES cell-mediated transgenesis has so far been limited to the mouse. In the fish medaka (Oryzias latipes) several ES cell lines have been made available. Here we report the optimized conditions for gene transfer and drug selection in the medaka ES cell line MES1 as a prelude for gene targeting in fish. MES1 cells gave rise to a moderate to high transfection efficiency by the calcium phosphate co-precipitation (5%), commercial reagents Fugene (11%), GeneJuice (21%) and electroporation (>30%). Transient gene transfer and CAT reporter assay revealed that several enhancers/promoters and their combinations including CMV, RSV and ST (the SV40 virus early gene enhancer linked to the thymidine kinase promoter) were suitable regulatory sequences to drive transgene expression in the MES1 cells. We show that neo, hyg or pac conferred resistance to G418, hygromycin or puromycin for positive selection, while the HSV-tk generated sensitivity to ganciclovir for negative selection. The positive-negative selection procedure that is widely used for gene targeting in mouse ES cells was found to be effective also in MES1 cells. Importantly, we demonstrate that MES1 cells after gene transfer and long-term drug selection retained the developmental pluripotency, as they were able to undergo induced differentiation in vitro and to contribute to various tissues and organs during chimeric embryogenesis.

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.

Transgene for growth hormone in common carp (Cyprinus carpio L.) promotes thymus development

The transgenic carp were produced by micro-injection of CAgcGHc into the fertilized eggs. Observation of the thymus development between the transgenics and non-transgenic controls was carried out. The thymus of one-year-old transgenics F1 showed a great increase in both size and weight. The unilateral thymus of the transgenics weighed from 190 to 295 mg with average 218.6 mg, whereas the unilateral thymus of the controls weighed 20-81 mg with average 42.5 mg; i.e. the thymus weight in the transgenics was 5.14 fold over that in the controls. The index of thymus/body weight in the transgenics was 2.97 fold over the controls. Light microscopy observation indicated that the thymus of the transgenics; well developed with the thickened outer region and compactly arranged thymocytes, while the thymus in the controls were degenerating with the thinned outer region, scattered thymocytes and groups of fatty cells. Further analysis with the electron microscopy revealed that pro-liferous cells in the transgenics; were mainly small lymphocytes and no pathological changes were found. The results confirmed that the "All-fish" GH-transgene promotes thymus development and thymocyte proliferation, and retards thymus degeneration. The study has laid a foundation for further analysis of the immunobiological function in GH-transgenic carp.

Agrobacterium-mediated transformation of Kentucky bluegrass

Embryogenic calli of Kentucky bluegrass, named Md, were induced from mature seeds and embryos, and proliferated on medium K3 containing 2,4-dichlorophenoxyacetic acid (2,4-D, 10.0 mumol/L), 6-benzylaminopurine (BAR, 0.5 mumol/L) and K5 which was the K3 medium supplemented with cupric sulfa (0.5 mumol/L) under dim-light condition (20-30 mumol.m(-2).s-1, 16 h light) at 24 degreesC. Embryogenic calli were transformed with plasmids pDM805 Carring bar and gus genes, Which was mediated by an Agrobacterium strain AGL1, four transgenic lines were obtained. The important factors that affect the transformation efficiency and obtain desirable number of transgenic plants included: (1) the quality of embryogenic calli; (2) light condition and time of co-cultivation; (3) concentration of antibiotics used for suppressing the overgrowth of Agrobacterium in the course of transformed plant regeneration; (4) selection pressure, etc. The micro nutrient of cupric had significant influence on the quality of embryogenic calli. This presentation is the first successful protocol of Kentucky bluegrass transformation mediated by Agrobacterium.

Development of a positive-negative selection procedure for gene targeting in fish cells

The gene targeting technique is a powerful tool for analyzing functions of cloned genes and for generating transgenic animals with site-directed integration of foreign genes. In order to develop this technique in fish, positive-negative selection (PNS) and homologous recombination vectors were constructed, and their expression was examined in fish cells. A vector (pNK) for PNS consists of the neomycin resistance gene (neo) as a positive selectable marker gene and the herpes simplex virus (HSV) thymidine kinase (tk) gene as a negative selectable marker gene. Positive selection with geneticin (G418) of epithelioma papulosum of carp (EPC) cells transfected with linearized pNK vector yielded 350 colonies, while double selection of transfected EPC cells with G418 and gancyclovir (Gc) resulted in nearly complete cell death, demonstrating that the PNS procedure is effective in fish cells. Homologous recombination vectors consist of the Xiphophorus melanoma receptor kinase (X mrk(Y)) gene as homologous sequence in addition to the neo and tk genes. Conditions for homologous recombination vector transfection and drug selection were established. After verification of the feasibility of expression of homologous recombination vectors in EPC cells, the first gene targeting experiments were attempted in the Xiphophorus melanoma cell line, PSM. Positive-negative selection of the targeting vector-transfectants led to a low enrichment in this particular cell line. The reasons for the low enrichment in PSM cells were discussed. (C) 2002 Elsevier Science B.V. All rights reserved.

Introduction of the human lactoferrin gene into grass carp (Ctenopharyngodon idellus) to increase resistance against GCH virus

Haemorrhage can be an epidemic and fatal condition in grass carp. It is known now that the Grass Carp Haemorrhage Virus (GCHV) triggers haemorrhage. Human lactoferrin (hLF) plays an important role in the non-specific immune system, making some organisms more resistant to some viruses. Sperm of grass carp was mixed with linearized pCAhLFc, which is a DNA construct containing an hLF cDNA and the promoter of common carp beta-actin gene, and then electroporated. Then, mature eggs were fertilized in vitro with the treated sperm cells. The fry were sampled and analyzed by polymerase chain reaction (PCR). Results indicated that the foreign gene had been transferred successfully into the cells of some fry. Under optimal electroporation conditions, the efficiency of gene transfer was as high as 46.8%. About 35.7% of treated 5-month-old grass carp contained foreign genes. Most transgenic fry demonstrated significant delays in onset of symptoms of haemerrhage after injection of GCHV, suggesting a significant positive relationship between hLF cDNA and levels of disease resistance (P < 0.01). Results suggest that transgenic grass carp could be bred for increased resistance to haemorrhage. (C) 2002 Elsevier Science B.V. All rights reserved.

The onset of foreign gene transcription in nuclear-transferred embryos of fish

The transcriptional onset of hGH-transgene in fish was studied in the following three cases: the first is in MThGH-transgenic F-4 common carp (Cyprinus carpio) embryos, the second is in nuclear-transferred embryos supported by the transgenic F-4 embryonic nuclei, and the third is in nuclear-transferred embryos supported by the transgenic F-4 tail-fin nuclei. RT-PCR results show that the hGH-transgene initiates its transcriptional activity from early-gastrula stage, the early blastula stage and even 16-cell stage in the first, second and third cases, respectively. it looks like that fish egg cytoplasm could just offer a very restricted reprogramming on transcriptional activity of specific gene in differentiated cell nuclei by nuclear transplantation.

Embryonic and genetic manipulation in fish

Fishes, the biggest and most diverse community in vertebrates are good experimental models for studies of cell and developmental biology by many favorable characteristics. Nuclear transplantation in fish has been thoroughly studied in China since 1960s. Fish nuclei of embryonic cells from different genera were transplanted into enucleated eggs generating nucleo-cytoplasmic hybrids of adults. Most importantly, nuclei of cultured goldfish kidney cells had been reprogrammed in enucleated eggs to support embryogenesis and ontogenesis of a fertile fish. This was the first case of cloned fish with somatic cells. Based on the technique of microinjection, recombinant MThGH gene has been transferred into fish eggs and the first batch of transgenic fish were produced in 1984. The behavior of foreign gene was characterized and the onset of the foreign gene replication occurred between the blastula to gastrula stages and random integration mainly occurred at later stages of embryogenesis. This eventually led to the transgenic mosaicism. The MThGH-transferred common carp enhanced growth rate by 2-4 times in the founder juveniles and doubled the body weight in the adults. The transgenic common carp were more efficient in utilizing dietary protein than the controls. An "all-fish" gene construct CAgcGH has been made by splicing the common carp beta-actin gene (CA) promoter onto the grass carp growth hormone gene (gcGH) coding sequence. The CAgcGH-transferred Yellow River Carp have also shown significantly fast-growth trait. Combination of techniques of fish cell culture, gene transformation with cultured cells and nuclear transplantation should be able to generate homogeneous strain of valuable transgenic fish to fulfil human requirement in 21(st) century.

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.

转座子Minos介导的果蝇转基因平台构建及水稻高表达启动子的克隆 任娟 指

本实验室果蝇研究工作，主要集中在黑腹果蝇的新基因起源的研究。新基因起源的分子机制主要包括：外显子重排、基因复制、基因逆转座、移动元件介导、基因水平转移、基因从头起源、基因的断裂融合。为了阐述这些新基因的产生和它们所带来的物种适应性，我们对这些新近起源的基因进行了功能研究。但是，仅仅限于新基因所在物种的功能研究并不能完全解释新基因产生的进化原因，我们需要了解它是否能够给没有该基因的果蝇物种带来一定的适应性。例如一些生殖相关新基因，如果我们将它们转入没有该基因的果蝇，那是否能够给该果蝇带来生殖能力的提高？无论结果如何，这都为我们研究新基因的起源提供一个重要线索。由此，黑腹果蝇以外的其它果蝇物种中实现转基因成为该研究的重要技术环节。但是，实验室目前的转基因系统仅限于P转座子介导的黑腹果蝇转基因系统，因而我们需要建立一种新的转基因平台。而转座子Minos打破物种范围的转基因特性，以及它的转座特点为我们提供了选择。转座子Minos是从果蝇D. hydei中克隆出来长约1.8kb的Ⅱ型转座子，Tc1家族转座元件成员。Minos的转座机制与大部分转座子一样，在宿主基因组里面实行着剪切和粘贴的运作机制。Minos在转座时，偏向插入TA位点并且主要集中于内含子区域，这样可以减少对插入位置基因的影响。此外，Minos在黑腹果蝇中的转座效率约30%，并且拥有一套成熟的选择标记。因此，Minos成为我们解决非黑腹果蝇转基因技术难题的首选。 在本文的工作中，我们采用由希腊Savakis教授（希腊分子生物学与生物技术研究所）提供的Minos转基因系统，完成果蝇的转基因实验。在这套转基因系统中，非自主的转座子Minos和转座酶基因被克隆到了不同载体当中。其中Minos转座子序列中插入了由3xP3眼睛特异表达的启动子介导表达的eGFP报告基因，而转座酶基因则由热激蛋白hsp70启动子调控表达。实验过程中，我们在果蝇D. melanogaster 和D. yakuba的胚胎中分别同时显微注射入含有转座子和转座酶本实验室果蝇研究工作，主要集中在黑腹果蝇的新基因起源的研究。新基因起源的分子机制主要包括：外显子重排、基因复制、基因逆转座、移动元件介导、基因水平转移、基因从头起源、基因的断裂融合。为了阐述这些新基因的产生和它们所带来的物种适应性，我们对这些新近起源的基因进行了功能研究。但是，仅仅限于新基因所在物种的功能研究并不能完全解释新基因产生的进化原因，我们需要了解它是否能够给没有该基因的果蝇物种带来一定的适应性。例如一些生殖相关新基因，如果我们将它们转入没有该基因的果蝇，那是否能够给该果蝇带来生殖能力的提高？无论结果如何，这都为我们研究新基因的起源提供一个重要线索。由此，黑腹果蝇以外的其它果蝇物种中实现转基因成为该研究的重要技术环节。但是，实验室目前的转基因系统仅限于P转座子介导的黑腹果蝇转基因系统，因而我们需要建立一种新的转基因平台。而转座子Minos打破物种范围的转基因特性，以及它的转座特点为我们提供了选择。转座子Minos是从果蝇D. hydei中克隆出来长约1.8kb的Ⅱ型转座子，Tc1家族转座元件成员。Minos的转座机制与大部分转座子一样，在宿主基因组里面实行着剪切和粘贴的运作机制。Minos在转座时，偏向插入TA位点并且主要集中于内含子区域，这样可以减少对插入位置基因的影响。此外，Minos在黑腹果蝇中的转座效率约30%，并且拥有一套成熟的选择标记。因此，Minos成为我们解决非黑腹果蝇转基因技术难题的首选。 在本文的工作中，我们采用由希腊Savakis教授（希腊分子生物学与生物技术研究所）提供的Minos转基因系统，完成果蝇的转基因实验。在这套转基因系统中，非自主的转座子Minos和转座酶基因被克隆到了不同载体当中。其中Minos转座子序列中插入了由3xP3眼睛特异表达的启动子介导表达的eGFP报告基因，而转座酶基因则由热激蛋白hsp70启动子调控表达。实验过程中，我们在果蝇D. melanogaster 和D. yakuba的胚胎中分别同时显微注射入含有转座子和转座酶所在的质粒。转座酶在37度条件诱导下进行表达，协助Minos完成转座过程。在转基因果蝇的阳性筛选中，我们利用眼睛特异表达的绿色荧光蛋作为选择标记。并且，我们通过PCR实验进一步验证了转基因果蝇的真实性。本研究中，我们对转基因实验条件进行了初步优化。我们通过对黑腹果蝇白眼突变品系W1118和D. yakuba注射后胚胎进行保湿，对D. yakuba注射胚胎进行非退壳处理。在改进条件下W1118和D. yakuba的存活率分别为10%和3%左右。通过筛选转基因阳性果蝇，我们得出Minos在W1118和D. yakuba中的转座效率分别在32%和20%左右。我们的实验结果再一次证实了Minos在果蝇D. melanogaster中可行性。同时，该工作也初步完成了在果蝇D. yakuba 中的第一次Minos介导的转基因实验，为新基因的跨物种功能研究奠定了实验基础。在未来的工作计划中，我们将采用Minos转基因系统，把实验室目前研究的黑腹果蝇新基因导入其它物种果蝇进行功能研究。 水稻是一种重要的世界粮食作物，世界上过半的人口以水稻为主食。水稻相对别的粮食作物来讲具有较小的基因组，并且拥有较好的基因组注释，是一种理想的单子叶模式生物。植物转基因技术的发展推动着水稻功能基因组学的研究，目前水稻的转基因技术主要依赖于土壤细菌农杆菌（Agrobacterium tumefaciens）T-DNA介导的外源基因染色体插入。在自然状态下，农杆菌的T-DNA位于Ti致瘤质粒当中。它包括了一些转座元件和一些帮助T-DNA转座的毒性蛋白基因和调节基因。由于Ti质粒上的T-DNA太长，并且没有太多的酶切位点，因此自然状态的T-DNA不适合进行转基因实验。为了方便T-DNA的实际应用，研究人员创立了双载体转基因系统。T-DNA转座区被分离到出Ti载体，并且装载到另外一个适合实验操作的质粒当中，而毒性蛋白表达基因等则保留在Ti质粒上。因此，在进行T-DNA介导的转基因实验时，需要同时存在T-DNA载体和Ti质粒。 本文以“水稻注释计划数据库RAP-DB”的表达数据为参考，选择了60个高表达基因的启动子区域进行克隆。通过对T-DNA载体pCAMBIA1301 进行改造，去掉其原来的35S启动子，将预测的基因启动子克隆到该载体中并与报告基 摘要 因GUS 基因融合。通过分子克隆实验，我们得到了45个高表达基因的启动子载体。最终，为了测试这45个启动子的启动效率，我们会将它们转化到水稻愈伤组织中通过启动子融合的GUS基于表达情况来判断我们启动子的启动效率。

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.

Effects of the “all-fish” GH (growth hormone) transgene expression on resistance to Ichthyophthirius multifiliis infections in common carp, Cyprinus carpio L.

A study was undertaken on the susceptibility of the F-4 generation of "all-fish" growth hormone transgenic carp, Cyprinus carpio L, against Ichthyophthirius multifiliis infections. When 1-year old, transgenic carp, with non-transgenic carp and non-manipulated carp (controls) were split into three batches, and experimental infections were performed throughout the 3-month period. All 72 fish were successfully infected. It was shown that there was a significant difference (P<0.01) on infection level between transgenics and non-transgenics, and transgenics and controls. It possibly resulted from transgenics that had stronger non-specific immune functions. In addition, fish surface area affected significantly infection level (P<0.001). Carp with larger surface area harboured more parasites for each type of fish, but transgenic with larger surface area than non-transgenics and controls (P<0.01), loaded fewer parasites than others. Besides, the time of infection also greatly influenced (P<0.001) infection level. Results showed that there was a significant decline in parasite infectivity through October to November (P<0.001). It was likely to suggest that there existed senescence resulted in failure of any I. multifiliis isolate maintenance. Significant difference in infectivity between isolate G from grass carp and isolate H from gold fish suggested that different parasite strains may exist. (C) 2009 Elsevier B.V. All rights reserved.