共查询到18条相似文献，搜索用时 434 毫秒
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本研究将100~300 pg含有肌肉特异表达启动子和绿色荧光蛋白(Green fluorescent protein,GFP)基因的重组质粒(smyd1:gfp)显微注射到大菱鲆(Scophthalmus maximus)受精卵动物极细胞中,通过细心培育,成功孵化出鱼苗约120尾。统计分析显示,显微注射后,大菱鲆胚胎存活率为4.8%。利用荧光显微镜观察大菱鲆胚胎及仔鱼,只在注射smyd1:gfp质粒的胚胎及仔鱼的肌肉中发现有绿色荧光。通过进一步PCR扩增检测,在注射的大菱鲆胚胎及仔鱼DNA中扩增出了GFP特异片段,大小约为340 bp。研究表明,本研究成功建立了大菱鲆显微注射技术,可为大菱鲆基因功能研究和遗传育种奠定基础。 相似文献
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草鱼呼肠孤病毒衣壳蛋白VP7基因真核表达载体pCI-VP7的构建及鉴定 总被引：1，自引：0，他引：1
将编码草鱼呼肠孤病毒(grass carp reovirus,GCRV)主要衣壳蛋白VP70.9kb的基因片段连接至克隆载体pMD19-T中,筛选阳性克隆并测序,经检测为正确序列后,再将目的片段克隆入真核表达载体pCI,筛选得到阳性重组质粒pCI-VP7。然后构建pCI-VP-GFP重组表达质粒(即GFP基因与VP7的一段上游基因融合表达),用PCR及酶切方法鉴定克隆的正确性。并用脂质体法将其转染入真核细胞COS-1和CIK进行瞬时表达,荧光显微镜观察及RT-PCR特异性检测。结果表明,GFP基因与VP7的一段上游基因被成功转染到COS-1和CIK细胞,并得到了很好的表达。进而证明pCI-VP7可以成功的表达,为GCRV基因疫苗的研制提供了实验资料。 相似文献
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为更好地开展鳜基因功能研究和药物筛选工作,提高基因转染效率,本研究以鳜囊胚期胚胎为材料,采用含有20%胎牛血清的DMEM培养基进行培养,建立了生长稳定的鳜胚胎细胞系MFE。在此基础上,采用绿色荧光蛋白(GFP)作为标记物,在HEK293T细胞中体外包装逆转录病毒,再感染MFE细胞系。MTT法分析表明传代后细胞培养96 h内,细胞生长率变化也经历增殖、降低到达稳定期。而且MFE细胞能稳定表达GFP基因,感染效率为20%±5%,而脂质体转染效率为3%±2%。可见包装病毒感染细胞不仅能获得稳转细胞系,效率也远高于脂质体瞬时转染。荧光定量PCR分析表明,MFE细胞系能表达Irf1、Irf3和Irf7基因,Irf1基因表达量最高。MFE细胞系受到poly I:C刺激后,Irf1、Irf3和Irf7的表达量分别升高3.5,2.3和2.1倍。因此,MFE细胞通过病毒感染可以获得较高的转染效率,该细胞可作为鳜免疫相关基因功能研究的工具。 相似文献
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为了探讨piggyBac转座子在鲤科鱼类及鳅科鱼类转基因中的适用性,实验通过将PAβ启动子控制的DsRed表达元件插入pigA3GFP中,构建转基因载体pigA3GFP-AβDsRed。用该载体对草鱼肾脏(CIK)细胞转染,结果显示,通过piggyBac转座子可将外源基因导入到CIK细胞的基因组中,来源于家蚕的A3启动子和来源于巨细胞病毒的CMV启动子在CIK细胞均具有活性。将转基因载体pigA3GFP、pigA3GFP-AβDsRed分别与辅助质粒helper-pigA3混合后以精子介导法导入金鱼和泥鳅的受精卵后,经荧光观察、PCR鉴定、Dot blotting鉴定,证实获得了转基因金鱼和转基因泥鳅。研究表明,PB转座子在鲤科鱼类及鳅科的一些鱼中具有转座活性。 相似文献
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传染性造血器官坏死病和传染性胰腺坏死病是以感染鲑科鱼类为主的两种传染病。为克隆传染性造血器官坏死病病毒IHNVG基因和传染性胰腺坏死病病毒IPNVVP2基因,并构建其重组腺病毒载体。利用RT-PCR方法分别扩增出IHNVG基因和IPNVVP2基因,通过多基因片段同源重组技术将IHNVG基因和IPNVVP2基因克隆到pAdTrack-CMV载体上,经过线性化后与pAdEasy-1载体在BJ5183菌体内同源重组,构建出重组腺病毒质粒,经PCR及NotⅠ和HindⅢ双酶切鉴定后,再经PacⅠ线性化后用于转染HEK-293细胞,获得重组腺病毒。通过绿色荧光蛋白(green fluorescent protein, GFP)的表达来监控重组腺病毒的复制情况,用western blot法分别检测糖蛋白(G蛋白)和VP2蛋白的表达,并测定重组病毒的滴度。结果显示,克隆出的IHNVG基因和IPNVVP2基因总长度为3 036 bp。该病毒在HEK-293细胞中分别表达出蛋白分子质量约为58 kD和54 kD的产物,其滴度为1.0×109.5 mL-1 TC... 相似文献
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半滑舌鳎vasa调控区的克隆、表达载体构建及其驱动活性检测 总被引：1，自引：1，他引：0
为研究半滑舌鳎(Cynoglossus semilaevis)vasa(Csvasa)基因调控区的功能,在已克隆的Csvasa基因编码序列的基础上,采用基因组步移和PCR扩增的方法克隆得到Csvasa调控区,通过生物信息学方法分析vasa基因5′区,并构建了含Csvasa基因调控区的绿色荧光蛋白(GFP)表达载体(p Csvasa-GFP-T),进一步通过显微注射技术初步验证调控区的驱动活性。结果表明,通过基因组步移和PCR扩增获得Csvasa 5′区5 166 bp和3′区1 655 bp,利用在线生物信息学软件对5′区序列进行分析,发现在转录起始点上游26 bp处存在保守的TATA框,以及潜在的转录因子结合点如SRY、Oct-1、Sox-5、CREB、GATA、AP-1、C/EBP、Sp-1、c-Myc、HNF、NKX2-5、V-Myb等。通过显微注射技术,将所构建的p Csvasa-GFP-T表达载体注射于青鳉(Oryzias latipes)受精卵并进行培养观测,发现Csvasa调控区能够驱动GFP在青鳉胚胎内表达,荧光表达率为81%。将有荧光的胚胎培养为成鱼,检测外源基因的整合率为11.5%。这些结果为进一步研究半滑舌鳎原始生殖细胞(PGCs)的标记、追踪和操作研究以及半滑舌鳎的性别控制等奠定了基础。 相似文献
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ABSTRACT: Green fluorescent protein ( GFP ) and red fluorescent protein ( RFP ) genes regulated by the medaka skeletal muscle actin promoter were microinjected into fertilized d-rR medaka eggs to establish transgenic medaka lines. Intense fluorescence was detected in skeletal muscle. During development, GFP and RFP became detectable in anterior somites at the 12- and 30-somete stages, respectively. After hatching, intense fluorescence in skeletal muscle enabled individual fish to be identified under normal lighting without fluorescent microscopy. Fluorescence was also observed in the gills and esophagus of the adult fish. These data indicated that medaka lines are convenient not only for the study of skeletal muscle but also for the identification of cells or individuals in various studies. 相似文献
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A marine fish cell line derived from the kidney of red-spotted grouper, Epinephelus akaara, designated as EAGK was established and characterized. The EAGK cells multiplied well in Leibovitz's L-15 medium containing 10% foetal bovine serum at 25 °C and have been subcultured for more than 90 passages. Karyotyping, chromosomal typing and ribosomal RNA (rRNA) genotyping analysis revealed that EAGK had a modal diploid chromosome number of 82 and was a fibroblast cell line originated from grouper. A severe cytopathic effect was observed in EAGK cells incubated with Singapore grouper iridovirus (SGIV), but not with soft-shelled turtle iridovirus, viral nervous necrosis virus or spring viraemia of carp virus. SGIV replication was further confirmed by immunofluorescence, electron microscopy and virus titre determination. Bright fluorescence was observed after transfection with fluorescent protein reporter plasmids, indicating that EAGK cells can be used to identify gene functions in vitro. In addition, the cell organelles including mitochondria and endoplasm reticulum changed and aggregated around virus factories after SGIV infection, suggested that the EAGK cell line could be an important tool for investigation of iridovirus-host interactions. 相似文献
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Jyh Yih Chen Chen Jian Chyi Lin Sheng Hwa Pan Chia Yu Kuo Ching Ming 《Fisheries Science》2006,72(5):1086-1095
The main function of the single whey acidic protein domain (SWD)-containing protein in shrimp is unknown. To elucidate the
function of the SWD-containing protein in vivo, the SWD-containing protein gene was isolated and characterized. A 9.3-kb shrimp SWD-containing protein gene, and a 3.9-kb
3′-flanking region. The shrimp SWD-containing protein gene contained three exons and two introns. Different fragments of the
shrimp SWD-containing protein 5′-flanking region were transfected into HeLa cells. The promoter activities were assayed by
basal human chorionic gonadotropin (HCG), luteinizing hormone-releasing hormone (LRH), and gonadotropin-releasing hormone
(GnRH) treatments. The in vitro actions of the SWD-containing protein promoter expression pattern were studied by transfection of an SWD-containing protein
promoter (1 kb)-driven green fluorescent protein (GFP) encoding the GFP cDNA transgene into the HeLa cell line, which was
then microinjected into zebrafish Danio rerio embryos. These results indicate that the shrimp SWD-containing protein promoter might play an important role in gene regulation
of sex hormones in mammalian cell lines and in gene regulation of developmental stages in zebrafish. 相似文献
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A new cell line was established from the heart of a cultured marine fish, half smooth tongue sole (Cynoglossus semilaevis), designated as CSH (Cynoglossus semilaevis heart cell line). The CSH cells grow over 400 days in minimum essential medium (MEM) supplemented with 10% fetal bovine serum
(FBS) and 2 ng/ml basic fibroblast growth factor (bFGF). The suitable temperature for the cell growth was 24–30°C with the
optimum growth at 24°C and a reduced growth at 12 and 30°C. FBS and bFGF concentration were the two important components for
CSH cells proliferation. Twenty percent FBS in the medium was found to be the optimum concentration and bFGF promoted the
growth of CSH cells. The double time of the cells at 24°C was determined to 73.39 h. Chromosome analysis revealed that 44%
of the cells maintained a normal diploid chromosome number (2n = 42) in the CSH cells at Passage 58. The fluorescent signals were observed in CSH after the cells were transfected with
green fluorescent protein (GFP) reporter plasmids. CSH cells showed the cytopathic effect (CPE) after infection with lymphosystis
disease virus (LCDV). Moreover, the LCDV particles can be observed in the cytoplasm of virus-infected cells by electron microscopy,
and a segment of MCP gene for major capsid protein of LCDV was found by PCR amplification DNA of virus-infected cells. 相似文献
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To monitor the probiotic Bacillus S11 (BS11) in vivo , wild-type cells were transformed with the green fluorescent protein (GFP)-expressing plasmid, pAD44-12, carrying the gfp mut3a gene under the constitutive Bacillus cereus UW85 promoter, and its non-GFP control plasmid pAD. Transformants with pAD44-12 (BS11-GFP), not pAD (BS11-pAD), expressed detectable but not too high levels of green fluorescence. Chloramphenicol resistance (BS11-GFP, BS11-pAD) and GFP fluorescence (BS11-GFP) as markers suggested that plasmid retention was 78–79% for both BS11-GFP and BS11-pAD cells after approximately 50 generations of growth without antibiotic selection. When mixed into shrimp feed at a final concentration ∼105 CFU g−1 , the inclusion of viable transformed bacteria in fed shrimps was observed. After feeding shrimp three times daily in 400-L cement tanks for 9 weeks, no significant differences in the average shrimp weight, the number of BS11 in either the culture water or in the shrimp's gut were seen between shrimp fed BS11-GFP and BS11-pAD or BS11, suggesting that expression of the gfp mut3a gene has no detectable effect on BS11 properties and shrimp growth. Histological examination of sections of shrimp's intestines following feeding with BS11-GFP demonstrated that BS11-GFP in shrimp feed survived and adhered onto the shrimp intestines' surface. BS11-GFP thus has good potential as a non-invasive marker tag for short-term experiments. 相似文献
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Tor tor is an important game and food fish of India with a distribution throughout Asia from the trans-Himalayan region to the Mekong River basin to Malaysia, Pakistan, Bangladesh and Indonesia. A new cell line named TTCF was developed from the caudal fin of T. tor for the first time. The cell line was optimally maintained at 28°C in Leibovitz-15 (L-15) medium supplemented with 20% fetal bovine serum (FBS). The propagation of TTCF cells showed a high plating efficiency of 63.00%. The cytogenetic analysis revealed a diploid count of 100 chromosomes at passage 15, 30, 45 and 60 passages. The viability of the TTCF cell line was found to be 72% after 6 months of cryopreservation in liquid nitrogen (-196°C). The origin of the cell lines was confirmed by the amplification of 578- and 655-bp sequences of 16S rRNA and cytochrome oxidase subunit I (COI) genes of mitochondrial DNA (mtDNA) respectively. TTCF cells were successfully transfected with green fluorescent protein (GFP) reporter plasmids. Further, immunocytochemistry studies confirm its fibroblastic morphology of cells. Genotoxicity assessment of H?O? in TTCF cell line revealed the utility of TTCF cell line as in vitro model for aquatic toxicological studies. 相似文献
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We discovered that some isolated eel skeletal muscle cells exhibited green fluorescence under a fluorescence stereomicroscope,
and we successfully isolated a novel fluorescent protein from the eel muscle homogenate. The protein was a monomer with a
molecular mass of 16.5–17 kDa and showed minor and major peaks at 280 and 493 nm, respectively, in the absorption spectrum.
The molar extinction coefficient at 493 nm was 41,300 M−1 cm−1 and A280/A493 was 0.083. Excitation and emission spectra of the protein showed maxima at 493 and 527 nm, respectively. Heat
treatment at 95°C for 10 min or 5% trichloroacetic acid treatment of the protein caused aggregation of the protein but did
not release any fluorescent components such as FAD into the supernatant after centrifugation. Fluorescence of the protein
remained after native PAGE, but not after SDS-PAGE. These results indicate that the purified fluorescent protein is not a
flavoprotein, and that its fluorescent chromophore is a covalently bound one, such as green fluorescent protein (GFP) from
jellyfish Aequoria victoria, but that its fluorescence requires its native conformation within the protein. Based on these results, we can conclude that
the fluorescent protein obtained from eel skeletal muscle is a novel GFP-like protein. 相似文献