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1.
Genetic analysis of the progeny of crosses involving strains of the mossPhyscomitrella patens obtained by re-transforming a stable transgenic line, indicates that the plasmid used for re-transformation inserts at or near the chromosomal location of the related plasmid used to obtain the original transgenic line. The resulting structure may be subject to gene silencing. 相似文献
2.
Uchino K Imamura M Shimizu K Kanda T Tamura T 《Molecular genetics and genomics : MGG》2007,277(3):213-220
We investigated the use of Minos as a vector for transgenesis in the silkworm, Bombyx mori. We first constructed a vector plasmid with the green fluorescent protein (GFP) gene fused with the silkworm cytoplasmic
actin gene (A3) promoter, and a helper plasmid with the Minos transposase gene controlled by the same A3 promoter. Injection of the vector and helper plasmid DNA into silkworm eggs produced
transgenic animals in the following generation. The efficiency of transgenic silkworm production using this method was much
lower than that obtained using piggyBac-mediated germ line transformation. However, >40-fold increase in the efficiency of producing transgenic silkworms was obtained
using an in vitro synthesized source of Minos transposase mRNA. We conclude that the Minos transposon is a useful vector for construction of transgenic silkworms, particularly when in vitro synthesized mRNA is used.
This is the first report showing that Minos can be used as a vector for germ-line transformation in lepidopteran insects. 相似文献
3.
The Hermes transposable element has been used to genetically transform a wide range of insect species, including the mosquito, Aedes aegypti, a vector of several important human pathogens. Hermes integrations into the mosquito germline are characterized by the non-canonical integration of the transposon and flanking
plasmid and, once integrated, Hermes is stable in the presence of its transposase. In an effort to improve the post-integration mobility of Hermes in the germline of Ae. aegypti, a transgenic helper Mos1 construct expressing Hermes transposase under the control of a testis-specific promoter was crossed to a separate transgenic strain containing a target
Hermes transposon. In less than 1% of the approximately 1,500 progeny from jumpstarter lines analyzed, evidence of putative Hermes germline remobilizations were detected. These recovered transposition events occur through an aberrant mechanism and provide
insight into the non-canonical cut-and-paste transposition of Hermes in the germ line of Ae. aegypti. 相似文献
4.
Characterization of rice transformed via an Agrobacterium-mediated inflorescence approach 总被引:4,自引:0,他引:4
Dong Jinjiang Kharb Pushpa Teng Weimin Hall Timothy C. 《Molecular breeding : new strategies in plant improvement》2001,7(3):187-194
Rice inflorescences were inoculated with Agrobacterium tumefaciens strain LBA4404 carrying plasmid pJD4 with application of vacuum infiltration. After co-cultivation, callus was initiated and subjected to hygromycin selection, and plants were regenerated from resistant callus lines. Based on the total number of co-cultivated inflorescences bearing flowers 1 to 3 mm in length, the average frequency for recovering independent transgenic rice plants was at least 30%. Seeds from selfed R0 plants were harvested within 6 months after initiation of the experiments. Genomic DNA blot analysis showed that genes in the T-DNA of the binary plasmid were stably integrated into the rice genome, typically at low copy number. In most, but not all, cases the transgene was transmitted to R1 progeny at a frequency characteristic for Mendelian inheritance of a single dominant trait. For selfed progeny of one two-locus insertion line, reactivation of GUS expression was observed for a single copy locus that segregated from a silenced multicopy locus. For this line and some additional plants, fluorescence in situ hybridization was used to visualize the chromosomal location of the transgene insert. 相似文献
5.
6.
I. V. Tanasienko A. I. Yemets Y. V. Pirko V. I. Korhkovyy N. Abumhadi Ya. B. Blume 《Cytology and Genetics》2011,45(1):1-6
The biolistic transformation method was used for genetic improvement of three commercial cultivars of barley (Oksamytoviy,
Vodogray, and Hetman). The plasmid pHLFTuBA containing target gene hLF encoding human lactoferrin under the control of the rice glutein B-1 promoter GluB-1 was used for transformation. The gene encoding mutant alfa-tubulin conferring resistance to trifluralin (dinitroaniline herbicide)
was used as the selective marker. The screening of different trifluralin concentrations ranging from 0.1–30 μM was used for
determination of selective concentration of the agent. Two transgenic barley lines of cultivars Oksamytoviy and Hetman’s callus
line were selected after 2–3 months of cultivation on 10 μM of trifluralin. To confirm stable integration of the transformed
gene, the PCR analysis of leafs from regenerated plant after their adaptation on the ground was carried out. The 734 bp fragment
of the target gene was amplified from both regenerated plants. 相似文献
7.
We have developed a new construct to generate transgenic mice with one plasmid that offers: (1) Cre/loxP-mediated spatial and temporally-controlled tissue-specific transgene expression; (2) A color-switching mechanism that uses spectrum-complementary genetically-encoded red (mRFP) and green (eGFP) fluorescent markers to label the transgene-expressing cells; (3) A bioluminescent marker that turns-on in the transgene-expressing cells; (4) eGFP as a cell surface marker in the transgene-expressing cells that facilitates the isolation and targeting of these cells. This vector was tested in vitro by co-transfection of the transgenic plasmid and a plasmid containing Cre recombinase into cultured cells and by establishing a transgenic mouse line. We show that this method allows versatile transgene expression targeting and color-switching to facilitate fluorescent and bioluminescent imaging both in cultured cells and in vivo. Our strategy provides time-saving features in tissue-specific transgene expression, bioimaging and primary cell isolation and can be used for generation of gene-specific transgenic mice. 相似文献
8.
Summary Fertile transgenic plants of the annual pasture legume Medicago truncatula were obtained by Agrobacterium-mediated transformation, utilising a disarmed Ti plasmid and a binary vector containing the kanamycin resistance gene under the control of the cauliflower mosaic virus 35S promoter. Factors contributing to the result included an improved plant regeneration protocol and the use of explants from a plant identified as possessing high regeneration capability from tissue culture. Genes present on the T-DNA of the Ri plasmid had a negative effect on somatic embryogenesis. Only tissue inoculated with Agrobacterium strains containing a disarmed Ti plasmid lacking the T-DNA region or a Ri plasmid with an inactivated rol A gene regenerated transgenic plants. Fertile transgenic plants were only obtained with disarmed A. tumefaciens, and the introduced NPT II gene was transmitted to R1 progeny.Abbreviations BAP
6-benzylaminopurine
- NAA
1-naphthaleneacetic acid
- NPT
neomycin phosphotransferase 相似文献
9.
Abul Mandal Mats Sandgren E. Tapio Palva 《In vitro cellular & developmental biology. Plant》1994,30(4):204-209
Summary Plasmid rescue can provide an efficient way of cloning T-DNA-tagged genomic DNA of plants. However, rescue has often been
hampered by extensive rearrangements in the cloned DNA. We have demonstrated using a transgenic line ofArabidopsis thaliana that the plant DNA flanking the T-DNA tag was heavily cytosine methylated. This methylation could be completely inhibited
by growing the plants in the presence of azacytidine. Rescue of the T-DNA tag together with the flanking plant genomic DNA
sequences from nontreated control plants into an modified cytosine restriction (mcr) proficient strain ofEscherichia coli resulted in rearrangements of the majority of the rescued plasmids. These rearrangements could be avoided if the methylation
was inhibited in the transgenic plants by azacytidine treatment or by cloning into anmcr-deficient strain ofE. coli. The results indicate that cytosine methylation of the DNA in the transgenic plants is the main cause of the DNA rearrangements
observed during plasmid rescue and suggest efficient strategies to eliminate such artifacts. 相似文献
10.
To study paraxial mesoderm formation in the mouse, transgenic lines that can be used to either selectively delete or express genes of interest in the paraxial mesoderm are required. We have generated a transgenic mouse line that expresses Cre recombinase in the paraxial mesoderm (PAM) beginning at e7.5. A lacZ Cre recombinase reporter line showed that in addition to PAM and its derivatives, lateral plate and intermediate mesoderm derivatives were also exposed to Cre activity, while the node, notochord, and cardiac mesoderm were not. We further demonstrate that 70–75% of the fibroblasts generated from Dll1‐msd Cre, ROSA26‐rtTA embryos possess Cre recombinase activity. These mice can therefore be used in combination with tet‐responsive transgenic lines to generate mesoderm‐derived embryonic fibroblasts that inducibly express a gene of interest. genesis 47:309–313, 2009. © 2009 Wiley‐Liss, Inc. 相似文献
11.
LIU Feng-Long ZHANG Hong-BinSHI Ding-Ji SHANG Zhi-Di LIN Chen SHAO Ning PENG GuohongZHANG Xue-Yan ZHANG Hai-Xia WU Jin-Yin XU Xu-Dong WANG JieJIANG Yue-Hua ZHONG Ze-PuZHAO Shu-JinWU Min CENG Cheng-Kui 《中国科学:生命科学英文版》1999,42(1):25-33
The construction of the shuttle, expression vector of human tumor necrosis factor alpha (hTNF-α) gene and its expression in
a cyanobacteriumAnabaena sp. PCC 7120 was reported. The 700-bp hTNF cDNA fragments have been recovered from plasmid pRL-rhTNF, then inserted downstream
of the promoter PpsbA in the plasmid pRL439. The resultant intermediary plasmid pRL-TC has further been combined with the
shuttle vector pDC-8 to get the shuttle, expression vector pDGTNF. The expression of the rhTNF gene inEscherichia coli has been analyzed by SDS-PAGE and thin-layer scanning, and the results show that the expressed TNF protein with these two
vectors is 16.9 percent (pRL-TC) and 15.0 percent (pDC-TNF) of the total proteins in the cells, respectively, while the expression
level of TNF gene in plasmid pRL-rhTNF is only 11.8 percent. Combined with the participation of the conjugal and helper plasmids,
pDC-TNF has been introduced intoAnabaena sp PCC 7120 by triparental conjugative transfer, and the stable transgenic strains have been obtained. The existence of the
introduced plasmid pDC-TNF in recombinant cyanobacterial cells has been demonstrated by the results of the agarose electrophoresis
with the extracted plasmid samples and Southern blotting with α-32P labeled hTNF cDNA probes, while the expression of the hTNF gene inAnabaena sp. PCC 7120 has been confirmed by the results of Western blotting with extracted protein samples and human TNF-alpha monoclonal
antibodies. The cytotoxicity assays using the mouse cancer cell line L929 proved the cytotoxicity of the TNF in the crude
extracts from the transgenic c~anobacteriumAnabaena sp. PCC 7120.
Project supported by the National Natural Science Foundation of China (Grant No. 39280016). 相似文献
12.
Transgenic hairy root system is important in several recalcitrant plants, where Agrobacterium tumefaciens-mediated plant transformation and generation of transgenic plants are problematic. Jute (Corchorus spp.), the major fibre crop in Indian subcontinent, is one of those recalcitrant plants where in vitro tissue culture has
provided a little success, and hence, Agrobacterium-mediated genetic transformation remains to be a challenging proposition in this crop. In the present work, a system of transgenic
hairy roots in Corchorus capsularis L. has been developed through genetic transformation by Agrobacterium rhizogenes harbouring two plasmids, i.e. the natural Ri plasmid and a recombinant binary vector derived from the disarmed Ti plasmid
of A. tumefaciens. Our findings indicate that the system is relatively easy to establish and reproducible. Molecular analysis of the independent
lines of transgenic hairy roots revealed the transfer of relevant transgenes from both the T-DNA parts into the plant genome,
indicating the co-transformation nature of the event. High level expression and activity of the gusA reporter gene advocate that the transgenic hairy root system, thus developed, could be applicable as gene expression system
in general and for root functional genomics in particular. Furthermore, these transgenic hairy roots can be used in future
as explants for plantlet regeneration to obtain stable transgenic jute plants. 相似文献
13.
Transgenic Zebrafish Expressing Chicken Lysozyme Show Resistance against Bacterial Diseases 总被引:2,自引:0,他引:2
We established a transgenic zebrafish strain expressing chicken lysozyme gene under the control of the Japanese flounder keratin
gene promoter, and investigated its resistance to a pathogenic bacterial infection. To generate the lysozyme transgenic construct,
Japanese flounder keratin promoter was linked to both the hen egg white (HEW) lyoszyme gene and green fluorescence protein
(GFP) gene used as a selection marker for the transgenic strains, in a recombinant plasmid. The recombinant plasmid was microinjected
into fertilized zebrafish eggs. In F2 transgenic zebrafish, GFP expression was strong in the epithelial tissues, liver and
gill from the embryonic stage to the adult stage. The expressions of HEW lysozyme and GFP mRNA were confirmed in the liver
and skin by RT-PCR. Western blot analysis showed that both HEW lysozyme and GFP were present in protein extracts from the
liver of transgenic zebrafish, but not in protein extracts from the muscle. The lytic activity of protein extracts from the
liver (assessed by a lysoplate assay using Micrococcus lysodeikticus as a substrate) was 1.75 times higher in F2 transgenic zebrafish than in the wild type. In a challenge experiment, 65% of
the F2 transgenic fish survived an infection of Flavobacterium columnare and 60% survived an infection of Edwardsiella tarda, whereas 100% of the control fish were killed by both pathogens. However, the survival rates of the transgenic fish were
not significantly higher when higher concentrations of bacteria were used. 相似文献
14.
‘Galia’ muskmelon (Cucumis melo L. var. reticulatus Ser.) has been recalcitrant to transformation by Agrobacterium tumefaciens. Transformation of the ‘Galia’ male parental line, ‘Krymka’, with an ACC oxidase (CMACO-1) gene in antisense orientation
is described herein. Explants were transformed using A. tumefaciens strain ABI, which contained a vector pCmACO1-AS plasmid, bearing an antisense gene of CMACO-1 and the CP4 syn gene (glyphosate-tolerance). Both CMACO-1 and CP4 syn genes were assessed by a polymerase chain reaction method. Flow cytometry analysis was performed to determine plant ploidy
level of primary transformants. Two completely diploid independent transgenic plants were obtained. Southern blot and segregation
analysis in the T1 generation determined that each independent transgenic line had one single insertion of the transgene. These transgenic muskmelon
male parental lines have potential for use in the production of ‘Galia’ F1 hybrids with improved shelf life. 相似文献
15.
Wagdy A. Sawahel 《Plant Molecular Biology Reporter》2001,19(4):377-377
Polybrene and/or spermidine treatments were used to deliver plasmid DNA into cotton suspension culture obtained from cotyledon-induced
callus. The transforming plasmid (pBI221.23) contained the selectablehpt gene for hygromycin resistance and the screenablegus gene. Primary transformant cotton plants were regenerated and analyzed by DNA hybridization and β-glucuronidase assay. The
combination polybrene-spermidine treatment greatly enhanced the uptake and expression of DNA and the recovery of nonchimeric
germ-line transgenic cotton plants. 相似文献
16.
17.
Z. Yin G.-L. Wang 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2000,100(3-4):461-470
The transfer of the long T-DNA (T-DNA and non-T-DNA) of a binary plasmid from Agrobacterium into the rice genome was investigated at both molecular and genetic levels. Out of 226 independent transgenic plants, 33%
of the transformants contained non-T-DNA sequences. There was no major difference in the frequency of non-T-DNA transfer among
three Agrobacterium tumefaciens strains.Four T1 plants containing a single putative long T-DNA insertion were selected for Southern analysis. Three of them
were confirmed to have a long T-DNA insertion with a size of greater-than-unit-length of the binary plasmid. This was further
confirmed by rescuing the intact binary plasmid from these plants. Our results suggest that long T-DNA transfer by rolling-circle
replication from Agrobacterium to rice occurs frequently, and that the high frequency of non-T-DNA transfer should be considered when producing transgenic
rice for commercial production.
Received: 22 April 1999 / Accepted: 22 June 1999 相似文献
18.
苏云金芽胞杆菌基因是转基因抗虫作物中通用的外源功能基因,在绝大多数抗虫转基因作物中均有存在,然而Bt基因检测标准样品的缺乏却限制了我国转Bt基因抗虫作物检测工作的发展。为了弥补传统基体标准样品的缺失,首先将Cry1Ab、Cry1Ac、Cry3A 3种常用Bt外源基因克隆到pUC57质粒上,通过测序、酶切和qPCR等技术对质粒的序列和扩增功能进行了验证,然后对扩增效率和实际应用情况加以测试,评价其转基因检测的适用性,构建了质粒标准分子。结果显示,制备的质粒标准分子测序结果与靶标序列完全符合,酶切结果、qPCR扩增结果和扩增效率等均符合预期,在Cry1Ab、Cry1Ac、Cry3A基因特异性检测中的应用符合阳性对照要求,表明制备的阳性质粒标准分子能够作为转Cry1Ab、Cry1Ac、Cry3A基因qPCR基因特异性检测的阳性标准样品。 相似文献
19.
E. B. Rukavtsova A. R. Gayazova E. N. Chebotareva Ya. I. Buryanov 《Russian Journal of Genetics》2009,45(8):924-928
The pBM plasmid, carrying the gene of hepatitis B virus surface antigen (HBsAg) and free of any selection markers of antibiotic or herbicide resistance, was constructed for genetic transformation of plants.
A method for screening transformed plant seedlings on nonselective media was developed. Enzyme immunoassay was used for selecting
transgenic plants with HBsAg gene among the produced regenerants; this method provides for a high sensitivity detection of HBsAg in plant extracts. Tobacco
and tomato transgenic lines synthesizing this antigen at a level of 0.01–0.05% of the total soluble protein were obtained.
The achieved level of HBsAg synthesis is sufficient for preclinical trials of the produced plants as a new generation safe
edible vaccine. The developed method for selecting transformants can be used for producing safe plants free of selection markers. 相似文献
20.
为了建立一种用于研究肌肉和心脏发育及其相关疾病的绿色荧光蛋白(enhanced green fluorescent protein,EGFP)转基因斑马鱼品系,本研究使用斑马鱼ttn.2基因编码区上游启动子序列和绿色荧光蛋白基因编码序列构建了重组表达载体,并将该载体和Tol2转座酶的加帽mRNA显微共注射入斑马鱼1-细胞期胚胎,通过荧光检测、遗传杂交筛选和分子鉴定等方法,成功建立了能稳定遗传的Tg(ttn.2:EGFP)转基因斑马鱼品系。荧光表达分析及原位杂交分析结果表明,绿色荧光信号在斑马鱼肌肉和心脏组织中特异表达模式与ttn.2基因的mRNA表达一致。通过反向PCR鉴定转基因表达载体在F1代斑马鱼品系中的随机整合位点,结果表明:No.33转基因品系的EGFP基因整合在斑马鱼的4号和11号染色体上,No.34转基因品系则整合在1号染色体上。该荧光转基因斑马鱼品系Tg(ttn.2:EGFP)的成功构建为肌肉和心脏发育以及相关疾病研究提供了一个新的理想实验模型。此外,绿色荧光强烈表达的斑马鱼品系还可以作为一种新的观赏鱼。 相似文献