Characterization and multi-generational stability of the growth hormone transgene (EO-1α) responsible for enhanced growth rates in Atlantic Salmon

Transgenic technologies provide a promising means by which desirable traits can be introduced into cultured fish species within a single generation thus accelerating the production of genetically superior broodstock for aquaculture. However, before such fish are allowed to be marketed as food they must receive government regulatory approval. Two pivotal regulatory requirements are: (1) complete characterization of the genomically integrated transgene and, (2) demonstration that the transgene remains stable over multiple generations. We have generated a stable line of growth hormone (GH) transgenic Atlantic salmon (Salmo salar) using an “all fish” gene construct (opAFP-GHc2) containing a growth hormone cDNA from chinook salmon whose expression is regulated by the 5′ promoter and 3′ termination regions derived from an ocean pout antifreeze protein (AFP) gene. In this study we show that a reorganized form of the opAFP-GHc2 construct (termed EO-1α) integrated as a single functional copy into a 35 bp repeat region of the genomic DNA. PCR based mapping revealed that the linear sequence of the EO-1α integrant was organized as follows: base pairs 1580–2193 of the ocean pout promoter region followed by the intact chinook salmon GH cDNA, the complete ocean pout antifreeze 3′ region, and the first 1678 bp of the ocean pout antifreeze 5′ region. Sequence analysis of the EO-1α integrant and genomic flanking regions in F₂ and F₄ generation salmon revealed that they were identical. In addition, apart from the disruption at the integration sites, the consensus sequences of the integrant in these two generations of salmon were identical to the sequence of the opAFP-GHc2 construct. These results indicate that the EO-1α transgene codes for the chinook salmon GH, and that the transgene and the integration site have remained stable over multiple generations.     

Antifreeze protein gene transfer in Atlantic salmon.

Salmonids freeze to death if they come into contact with ice. Many marine fish species that inhabit icy sea waters synthesize antifreeze proteins (AFP) to protect them from freezing. Production of stable lines of freeze-resistant salmon and other species would greatly facilitate development of sea-pen aquaculture in many regions. We successfully introduced winter flounder AFP genes into Atlantic salmon. Research to date indicates stable genomic integration and low levels of expression of winter flounder AFP genes in a small number (approximately 3%) of salmon developed from microinjected eggs. Inheritance of the AFP gene by offspring (F1) from crosses between transgenic and wild-type salmon revealed that the transgenic flounders (F0) were germ-line mosaics. Low levels of AFP precursors could be detected in the blood of all these transgenic offspring (F1). Approximately 50% of the progeny produced by crosses between transgenic F1 and wild-types contained the AFP genes. These results demonstrate that stable germ-line transformed Atlantic salmon can be produced.     

Tissue specific expression of antifreeze protein and growth hormone transgenes driven by the ocean pout (<Emphasis Type="Italic">Macrozoarces americanus</Emphasis>) antifreeze protein OP5a gene promoter in Atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis>)

Previous research aimed at producing genetically improved salmon broodstock for aquaculture led to the creation of two lines of transgenic Atlantic salmon using gene constructs that were derived in part from the ocean pout OP5a antifreeze protein (AFP) gene. One of the lines was produced using an OP5a AFP gene in which the 5′ region of the promoter was removed (termed t-OP5a-AFP), and the other line contains a growth hormone (GH) transgene (EO-1α) that consists of a chinook salmon GH cDNA driven by a truncated OP5a AFP promoter that is almost identical to that of the t-OP5a-AFP construct. The similarity of the promoter regions of these transgenes provided an opportunity to evaluate their tissue specific expression patterns. Expression of mRNA was evaluated using Northern blot and RT-PCR techniques. The results demonstrate that the AFP and GH trangenes were expressed in almost all body tissues, suggesting that the promoter region of the OP5a AFP gene lacks tissue specific elements. Northern analysis revealed that expression of the t-OP5a-AFP gene was considerably greater than that of the EO-1α GH transgene. Only the spleen tissue of the GH transgenics showed a visible band of hybridization. In contrast clear bands of hybridization were evident in all tissues, except for blood cells, of the AFP transgenics with heart, liver and brain tissue showing the highest levels of mRNA expression. This higher level of expression could be attributable to the presence of introns in the t-OP5a-AFP transgene. Since the GH transgenic salmon grow considerably faster than non-transgenics the low levels of GH transgene expression in this line were clearly sufficient to produce the desired rapid growth phenotype. In contrast the levels of AFP expression were inadequate to impart any improvement in the freeze resistance of the AFP transgenic salmon.     

Effective generation of transgenic pigs and mice by linker based sperm-mediated gene transfer.

Background

Transgenic animals have become valuable tools for both research and applied purposes. The current method of gene transfer, microinjection, which is widely used in transgenic mouse production, has only had limited success in producing transgenic animals of larger or higher species. Here, we report a linker based sperm-mediated gene transfer method (LB-SMGT) that greatly improves the production efficiency of large transgenic animals.

Results

The linker protein, a monoclonal antibody (mAb C), is reactive to a surface antigen on sperm of all tested species including pig, mouse, chicken, cow, goat, sheep, and human. mAb C is a basic protein that binds to DNA through ionic interaction allowing exogenous DNA to be linked specifically to sperm. After fertilization of the egg, the DNA is shown to be successfully integrated into the genome of viable pig and mouse offspring with germ-line transfer to the F1 generation at a highly efficient rate: 37.5% of pigs and 33% of mice. The integration is demonstrated again by FISH analysis and F2 transmission in pigs. Furthermore, expression of the transgene is demonstrated in 61% (35/57) of transgenic pigs (F0 generation).

Conclusions

Our data suggests that LB-SMGT could be used to generate transgenic animals efficiently in many different species.     

血管内皮生长因子 (VEGF1 83)转基因小鼠的建立(英文)

采用显微注射法 ,将一个由 1.3kb人感光细胞间维生素A类结合蛋白 (hIRBP)启动子区和人血管内皮生长因子 (hVEGF183)cDNA所构成的融合基因片段HIRV183导入 2 89枚ICR小鼠受精卵的雄性原核 .制备当代 (G0 )转基因小鼠 ,共得G0 代小鼠 17只 .经PCR和PCR Southern方法筛选得到 1只为hVEGF183转基因阳性小鼠 ,整合率为 5 9%,转基因总效率为 0 3%.将其与正常ICR小鼠交配 ,经PCR和dot blot杂交鉴定后代 (F1)小鼠阳性整合率为 4 5 9%.F1代兄妹之间进行交配 ,后代 (F2 )小鼠阳性整合率为 6 7 6 %.结果表明 ,外源基因hVEGF183整合在小鼠染色体的单一位点 ,为以后利用转基因动物的方法对VEGF这个新型异构体VEGF183进行深入研究打下了基础     

Stable Long Term Germ-Line Transmission of Transgene Integration Sites in Mice

Transgenic mice provide a valuable tool in all fields of basic and applied biological and medical research. In this study, we describe the fate of integrated transgenes in the mammalian host genome over a large number of generations. The stability of the germ-line transmission of integrated tyrosinase transgene copies was monitored up to generation F20 in a large number of individuals from seven transgenic mouse lines. Phenotypic and molecular genetic analysis of the offspring both within the different lines and in cross-breeding experiments revealed the high stability of the transgene integration sites in mice. Only very few individuals were affected by a transgene copy loss. These results indicate that, once homozygous transgenic lines are established, breeding programs can be continued to a high number of generations without further stringent molecular genetic analysis.     

A transgene and its expression profile are stably transmitted to offspring in transgenic medaka generated by the particle gun method

A particle gun is used in a potential method for introducing foreign genes into fish. In this paper, we report on the stable transmission of a transgene and its expression profile of the F4 generation in the transgenic medaka (Oryzias latipes). We established four transgenic strains, which contained a green fluorescent protein (GFP) gene controlled by a medaka beta-actin promoter, using a particle gun. One more transgenic strain was also generated by microinjection for comparison. In all five strains, the founder was discovered to be mosaic for the transgene. However, from the F1 to F4 generations, transgenes and their expression profiles were stably inherited in the Mendelian manner. The expression profile was common among the five strains regardless of the method for gene transfer: GFP fluorescence became detectable at an early neurula stage. In this stage, the fluorescence was observed ubiquitously in most tissues. As somite developed, GFP fluorescence became intense only in the skeletal muscle and lens but it decreased in other tissues. In adult fish, an intense fluorescence was restricted in the skeletal muscle and lens, while a considerably weak fluorescence was observed in the brain, gill, heart, kidney, spleen, and ovary. From these results, it was concluded that the transgene and its expression profile were stably transmitted to offspring, and thus the particle gun is an effective method for transgenesis in spite of its easiness.     

A Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the C. elegans Genome

Microinjecting DNA into the cytoplasm of the syncytial gonad of Caenorhabditis elegans is the main technique used to establish transgenic lines that exhibit partial and variable transmission rates of extrachromosomal arrays to the next generation. In addition, transgenic animals are mosaic and express the transgene in a variable number of cells. Extrachromosomal arrays can be integrated into the C. elegans genome using UV irradiation to establish nonmosaic transgenic strains with 100% transmission rate of the transgene. To that extent, F1 progenies of UV irradiated transgenic animals are screened for animals carrying a heterozygous integration of the transgene, which leads to a 75% Mendelian transmission rate to the F2 progeny. One of the challenges of this method is to distinguish between the percentage of transgene transmission in a population before (X% transgenic animals) and after integration (≥75% transgenic F2 animals). Thus, this method requires choosing a nonintegrated transgenic line with a percentage of transgenic animals that is significantly lower than the Mendelian segregation of 75%. Consequently, nonintegrated transgenic lines with an extrachromosomal array transmission rate to the next generation ≤60% are usually preferred for integration, and transgene integration in highly transmitting strains is difficult. Here we show that the efficiency of extrachromosomal arrays integration into the genome is increased when using highly transmitting transgenic lines (≥80%). The described protocol allows for easy selection of several independent lines with homozygous transgene integration into the genome after UV irradiation of transgenic worms exhibiting a high rate of extrachromosomal array transmission. Furthermore, this method is quite fast and low material consuming. The possibility of rapidly generating different lines that express a particular integrated transgene is of great interest for studies focusing on gene expression pattern and regulation, protein localization, and overexpression, as well as for the development of subcellular markers.     

Gene transfer into zebrafish by sperm nuclear transplantation

A technique for fertilizing zebrafish eggs by injection of sperm nuclei is described. Eggs that cleave normally can develop into swimming larvae and give rise to fertile adults. If sperm nuclei are preincubated for 20 min with DNA encoding the green fluorescent protein, transgene expression can be detected in all cells of the embryo. The use of condensed sperm nuclei allows injection with a small bore pipette, which is critical for successful injection of the relatively small zebrafish egg. This technique enables the generation of ubiquitously expressing transgenic zebrafish directly by microinjection. Hence, experiments involving transgenic fish can be completed in days, without the need for growing and breeding founders. This technique may also be used to generate transgenic lines, as transgene expression was visible in the offspring of transgenic founders. The method described here is likely to be applicable to other teleosts, such as medaka and salmon.     

Generation of Ci-Brachyury-GFP stable transgenic lines in the ascidian Ciona savignyi

We report generation of stable transgenic lines of the ascidian Ciona savignyi carrying a Ciona intestinalis-Brachyury-promoter/Green Fluorescent Protein-reporter (Ci-Bra-GFP) construct. The transgenic lines were made using a technique in which the endonuclease I-SceI was coinjected into fertilized eggs with a transgene construct containing flanking recognition sites for I-SceI. Two founder animals, out of 12 F(0) adults tested, were found to transmit the transgene to their offspring (F(1)s) at frequencies of 42% and 23%. The transgene was further inherited by the F(2) in a Mendelian fashion and displayed nonmosaic expression, indicating integration into the genome. The Mendelian inheritance and the absence of mosaicism persisted through the F(3) and F(4) generations. Southern blot analyses showed that the transgene was organized in tandem arrays of no more than 10 copies. Using these Ci-Bra-GFP transgenics, we describe cellular movements and shape changes involved in notochord morphogenesis in both wildtype and mutant embryos.     

人G-CSF转基因鼠的稳定遗传与表达

利用人粒细胞集落刺激因子（Ｇ－ＣＳＦ）基因组基因作为目的片段,将其受控于２．６ｋｂ的小鼠乳清酸蛋白（ＷＡＰ）基因的调控区下,通过显微注射法获得了两只整合有人Ｇ－ＣＳＦ转基因小鼠,通过繁殖建立了稳定的转基因系．一些表型参数测定表明转基因鼠与正常鼠无明显差别．通过ＲＴ－ＰＣＲ及Ｓｏｕｔｈｅｒｎｂｌｏｔ检测,在乳腺表达出人Ｇ－ＣＳＦ,为乳腺表达外源蛋白质及今后大动物研究奠定了基础．     

Generation of chickens expressing Cre recombinase

Cre recombinase has been extensively used for genome engineering in transgenic mice yet its use in other species has been more limited. Here we describe the generation of transgenic chickens expressing Cre recombinase. Green fluorescent protein (GFP)-positive chicken primordial germ cells were stably transfected with β-actin-Cre-recombinase using phiC31 integrase and transgenic chickens were generated. Cre recombinase activity was verified by mating Cre birds to birds carrying a floxed transgene. Floxed sequences were only excised in offspring from roosters that inherited the Cre recombinase but were excised in all offspring from hens carrying the Cre recombinase irrespective of the presence of the Cre transgene. The Cre recombinase transgenic birds were healthy and reproductively normal. The Cre and GFP genes in two of the lines were closely linked whereas the genes segregated independently in a third line. These founders allowed development of GFP-expressing and non-GFP-expressing Cre recombinase lines. These lines of birds create a myriad of opportunities to study developmentally-regulated and tissue-specific expression of transgenes in chickens.     

Production of offspring from cloned transgenic RFP female dogs and stable generational transmission of the RFP gene

The purpose of this study was to analyze the reproductive ability of transgenic female dogs born bysomatic cell nuclear transfer and to determine inheritance of the red fluorescent protein (RFP) transgene. The four founder transgenic bitches (F0) reached puberty at 340.8 ± 39.6 days after birth and were bred with wild-type male dogs by natural mating or by artificial insemination. The bitches all became pregnant and successfully delivered 13 puppies (F1), of which two females were bred with wild-type dogs to deliver 7 offspring (F2), including 1 stillbirth. Among the 19 live offspring, 10 puppies showed emission of RFP under UV light and the presence of the RFP transgene was confirmed by genomic PCR and Southern blot analyses. In conclusion, transgenic RFP female dogs exhibited normal reproductive ability and expression of the transgene was demonstrated in F1 and F2 generations.     

Spontaneous germline amplification and translocation of a transgene array.

The majority of the mammalian genome is thought to be relatively stable throughout and between generations. There are no developmentally programmed gene amplifications as seen in lower eukaryotes and prokaryotes, however a number of unscheduled gene amplifications have been documented. Apart from expansion of trinucleotide repeats and minisatellite DNA, which involve small DNA elements, other cases of gene or DNA amplifications in mammalian systems have been reported in tumor samples or permanent cell lines. The mechanisms underlying these amplifications remain unknown. Here, we report a spontaneous transgene amplification through the male germline which resulted in silencing of transgene expression. During routine screening one mouse, phenotypically negative for transgene expression, was found to have a transgene copy number much greater than that of the transgenic parent. Analysis of the transgene expansion revealed that the amplification in the new high copy transgenic line resulted in a copy number approximately 40-60 times the primary transgenic line copy number of 5-8 copies per haploid genome. Genetic breeding analysis suggested that this amplification was the result of insertion at only one integration site, that it was stable for at least two generations and that the site of insertion was different from the site at which the original 5-8 copy array had integrated. FISH analysis revealed that the new high copy array was on chromosome 7 F3/4 whereas the original low copy transgene array had been localised to chromosome 3E3. DNA methylation analysis revealed that the high copy transgene array was heavily methylated. The amplification of transgenes, although a rare event, may give insight into amplification of endogenous genes which can be associated with human disease.     

应用荧光原位杂交检测人凝血因子Ⅸ在转基因小鼠染色体上的整合

应用荧光原位杂交（FISH）技术检测两个转基因小鼠家系从F1到F4代的整合情况。阳性转基因小鼠98%~100%的中期分裂相,85%~94%的间期核出现杂交信号;阴性对照小鼠100%的中期分裂相、95%~96%的间期核未出现杂交信号。结果表明,该FISH实验条件能对转基因整合位点进行高效特异检测。本文分析的两家系转基因小鼠均为单位点整合, 但整合位点不同。各家系内F1到F4代的转基因小鼠均可检出整合染色体,且整合位点相同,表明外源基因稳定整合并遗传给后代。 Abstract:Fluorescence in situ hybridization （FISH） was used to detect the integration of hFⅨ on chromosomes of transgenic mice from F1 to F4 generation in two strains.For transgenic mice,98%~100% of metaphases and 85%~94% of interphases showed hybridization signal.For negative control mice,100% of metaphases and 95%~96% of interphases showed no hybridization signal.The results demonstrated that FISH developed to detect the integration sites of hFⅨ was high efficient and specific.The integration sites of the transgenic mice analyzed were both single but different between the two strains.The integration chromosomes can be found in the transgenic mice from F1 to F4 generation and the integration sites were the same as each of the strains,which indicated that the transgene was stably integrated and transmitted to offspring.