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1.
A fusion plasmid, pMK, containing the mouse metallothionein-I promoter/regulatory region joined to the structural gene of herpesvirus thymidine kinase, was introduced into mice by microinjection into fertilized eggs followed by reinsertion of the eggs into foster mothers. Fifteen percent (10 of 69) of the mice developing from this procedure carried pMK sequences. Seven of these mice expressed high levels of viral thymidine kinase in the liver. This enzyme is inducible by heavy metals, as indicated by assay of thymidine kinase activity following sequential partial hepatectomies with or without cadmium treatment. However, glucocorticoid treatment has been ineffective in all transgenic mice tested. The pMK sequences are extensively methylated at a variety of restriction sites, indicating the existence of a de novo methylation enzyme. We have analyzed the inheritance of pMK sequences and their expression in several pedigrees. These fusion genes are inherited as though they were integrated into a single chromosome; however, their expression may be extinguished, diminished or enhanced in the offspring relative to that of the parent. In some animals there is a correlation between changes in DNA methylation and expression of these fusion genes. 相似文献
2.
Expression of a foreign gene in a line of transgenic mice is modulated by a chromosomal position effect. 总被引:23,自引:4,他引:19
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Unusual aberrant expression of a foreign gene in a particular transgenic mouse line is often attributed to chromosomal position effect, although proof of this is lacking. An alternative explanation is that expression has been modified by the arrangement of multiple copies of the foreign gene at the insertion site or by mutation or gene rearrangement. We have distinguished between these explanations in the case of one particular transgenic line by recovering the aberrantly expressed foreign DNA and reintroducing it into the mouse genome to produce secondary transgenic mice. The expression pattern of the gene in the secondary transgenic mice was normal, showing that this case of aberrant expression is due to a chromosomal position effect. 相似文献
3.
Analysis of the integrant in MyK-103 transgenic mice in which males fail to transmit the integrant. 总被引:9,自引:5,他引:4
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Males in the transgenic mouse pedigree MyK-103, although fertile, do not transmit the integrant to offspring. The integrant is on chromosome 6 near the T-cell receptor beta-chain locus. It contains four fragments of the plasmid pMK (a metallothionein-thymidine kinase fusion gene) and a 532-base-pair fragment of displaced mouse DNA originating from a previously uncharacterized repetitive DNA family. The integration complex is flanked on either side by a 5-kilobase duplication of mouse DNA normally found in a single copy at this locus. Sequence analysis of the six novel junctions and their donor sequences shows that plasmid-plasmid junctions occurred at patches of limited homology, whereas chromosome-plasmid junctions were nonhomologous. 相似文献
4.
Hao Chen Yanhui Yin Yani Zhang Min Sun Tingfeng Chen Caiye Zhu Wei Li Bichun Li Huilin Yang 《Molecular biology reports》2013,40(3):2215-2222
The objective of this study was to explore the possibility of obtaining stable transgenic animals by intratesticular injection. The recombinant vector pEGFP-H-FABP expressing the goat heart-type fatty acid binding protein and green fluorescent protein was mixed with liposome complexes and randomly injected into the testes of mice. Testicular section, fluorescence, and DNA detection assays of mouse sperm were performed to determine the integration of foreign DNA. The results showed that foreign DNA was successfully expressed in the treated mice. Furthermore, the expression and function of the foreign gene were analyzed in F1 generation and F2 generation mice at different levels, with the positive rates of foreign gene transfer into the F1 and F2 generations being 4.0 and 30.23 %, respectively. These results strongly support testicular injection as an effective method of producing transgenic animals and indicate that foreign genes can be stably passed on to the offspring. This research has theoretical and practical implications for the improvement in the quality of laboratory animals and for gene therapy. 相似文献
5.
本工作构建了含有hDAF基因的转基因小鼠,以便研究hDAF基因能否消除异种器官移植中的排斥反应。 采用DNA重组的方法构建hDAF基因的表达载体pSP64HP(Fig.1)。通过受精卵显微注射,将其中的目的基因片段,转移到小鼠体内,建立转基因小鼠。再通过Dot blotting和Southern blotting杂交方法对出生小鼠的基因组特征进行查证。 连续两次对直接裂解菌液做PCR扩增,筛选出重组质粒(Fig.2&3),酶切图谱(Fig.4)和Southern杂交(Fig.5)分析结果与预期吻合,出现预期条带;小鼠受精卵注射后存活比率为77.9%,受精卵的发育率为3.4%,出生小鼠中,10.5%出现清晰杂交信号。 表明:hDAF基因表达载体构建成功;并整合入小鼠基因组中。 相似文献
6.
degQ基因编码一个由46个氨基酸组成的多肽,能增强许多芽孢杆菌胞外酶基因的表达.以pMK4作克隆载体构建短小芽孢杆菌基因文库,并用DNA探针原位杂交法从中钓出degQ基因.对克隆基因的DNA序列进行了分析并证明克隆的短小芽孢杆菌degQ基因具有增强枯草杆菌蛋白酶和果聚糖蔗糖酶基因表达的能力.degQ基因克隆有助于研究芽孢杆菌的正调控机理并可望提高外源基因在芽孢杆菌中表达. 相似文献
7.
《Genetic analysis, techniques and applications》1994,11(5-6):158-164
Because the P1 bacteriophage packages DNA inserts of 80–100 kb, which are much larger than inserts of bacteriophage λ or cosmid vectors, P1 DNA can be used to express large genes in cultured cells and transgenic mice. We obtained a P1 bacteriophage clone with a 79.5-kb insert (p158) that spanned the entire human apolipoprotein (apo-) B gene. We used the insert from p158 to express the human apo-B gene in both cultured rat hepatoma cells and transgenic mice. In this article, we review our apo-B expression studies and discuss the techniques that we have used for these expression studies. 相似文献
8.
红系特异的GFP基因在转基因小鼠中的整合和表达 总被引:4,自引:0,他引:4
应用荧光定量PCR技术对由位点控制区LCR的HS2元件和 β 珠蛋白基因启动子指导的红系特异表达绿色荧光蛋白 (GFP)基因的转基因小鼠中外源基因拷贝数进行测定 ,使用荧光显微镜和流式细胞仪检测小鼠外周血中GFP的表达水平 ,并运用荧光原位杂交技术 (FISH)确定了其中两只转基因小鼠中外源基因的整合位点 ,结果表明 :在转基因小鼠中外源基因的拷贝数各不相同且相差较大 ,而且拷贝数与GFP基因的表达量之间未呈现出相关性 ;FISH分析确定出两只转基因小鼠的外源基因整合于不同的染色体上 ;杂交信号的强弱与拷贝数的多少相一致 相似文献
9.
Tacken Paul J. Zee Andre van der Beumer Tim L. Florijn Ralph J. Gijpels Marion J. J. Havekes Louis M. Frants Rune R. Dijk Ko Willems van Hofker Marten H. 《Transgenic research》2001,10(3):211-221
The generation of functional transgenes via microinjection of overlapping DNA fragments has previously been reported to be successful, but it is still not a widely applied approach. Here we show that the method is very reliable, and should be considered, in case a single large insert clone of the desired gene is not available. In the present study, two large DNA fragments consisting of overlapping cosmids, together constituting the human very low density lipoprotein receptor (VLDLR) gene (35kb), were used to generate VLDLR transgenic (VLDLR-Tg) mice. Three transgenic founders were born, of which two (strain #2 and #3) generated transgenic offspring. Using Fiber-FISH analysis, the integration site was shown to contain at least 44 and 64 DNA fragments in mouse strains #2 and #3, respectively. This copy number resulted in integration sites of 1.5 and 2.5 megabase in size. Notably, over 90% of the fragments in both mouse strains #2 and #3 were flanked by their complementary fragment. In line with this observation, Southern blot analysis demonstrated that the correct recombination between fragments predominated in the transgenic insertion. Human VLDLR expression was detected in testis, kidney and brain of both mouse strains. Since this pattern did not parallel the endogenous VLDLR expression, some crucial regulatory elements were probably not present in the cosmid clones. Human VLDLR expression in testis was detected in germ cells up to the meiotic stage by in situ mRNA analysis. Remarkably, in the F1 generation of both VLDLR-Tg mouse strains the testis was atrophic and giant cells were detected in the semineferous tubuli. Furthermore, male VLDLR-Tg mice transmitted the transgene to their progeny with low frequencies. This could imply that VLDLR overexpression in the germ cells disturbed spermatogenesis. 相似文献
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转基因小鼠中外源基因遗传及表达稳定性的研究 总被引:2,自引:0,他引:2
挑选两个乳汁中人凝血因子IX(hFIX)表达量相差较大的转基因小鼠家系,分别用PCR、Southern blot、FISH和ELISA对两个家系中的小鼠进行检测。结果显示后代小鼠的转基因阳性率为50%左右;外源基因的整合是完整的,没有发现可见的丢失现象;家系中的各个小鼠表达量有差异,FIX-33家系中hFIX在乳汁中的表达量为(43.32±5.41)?g/mL;FIX-124家系中hFIX在乳汁中的表达量是(1.16±0.45)?g/mL。而两个家系之间的表达量则差异极为显著(P<0.01)。这表明原代转基因小鼠的遗传及表达特性可以得到稳定的传递。 相似文献
13.
Suganuma R Pelczar P Spetz JF Hohn B Yanagimachi R Moisyadi S 《Biology of reproduction》2005,73(6):1157-1163
We have developed a novel method for mouse transgenesis. The procedure relies on a hyperactive Tn5 transposase to insert a transgene into mouse chromosomes during intracytoplasmic sperm injection. This procedure integrates foreign DNA into the mouse genome with dramatically increased effectiveness as compared to conventional methods such as pronuclear microinjection and traditional sperm injection-mediated transgenesis. Our data indicate that with this method, transgenic mice, both hybrids and inbreds, can be produced more consistently and with lower numbers of manipulated oocytes required for traditional microinjection methods. The transposase-mediated transgenesis technique is also effective with round spermatids, offering the potential for rescuing the fertility of azoospermic animals using sperm precursor cells. 相似文献
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The ability to introduce cloned genes into mouse germ line has been used for analyzing cis-acting DNA sequences involved in tissue-specific and developmental regulation of the introduced gene. Using this system we have attempted to produce a transgenic mouse model for human dominantly inherited disease, familial amyloidotic polyneuropathy. Recently the mutant transthyretin gene which is considered to be responsible for this disease has been cloned and well characterized at molecular level. We have produced transgenic mice by microinjecting human mutant gene. Amyloid deposition was observed in the mucosa of alimentary tract and renal glomeruli, suggesting that this approach is successful in establishing the mouse model for human genetic disease. In addition, these experiments suggest that the expression of the mutant gene is regulated normally during developmental process and that the cause of adult onset is not due to the dysregulation of this gene expression. 相似文献
16.
We have achieved high-efficiency uptake and expression of foreign DNA in mouse Ltk- cells by modifying the DEAE-dextran-mediated transfection method of McCutchan and Pagano (J. Natl. Cancer Inst. 42:351-357, 1968) to include an initial incubation at elevated pH followed by a shock treatment with dimethyl sulfoxide. Up to 80% of mouse Ltk- cells transfected with the herpes simplex virus thymidine kinase gene expressed thymidine kinase as measured by autoradiography. 相似文献
17.
Rival-Gervier S Viglietta C Maeder C Attal J Houdebine LM 《Molecular reproduction and development》2002,63(2):161-167
Silencing of transgenes is a frequent event after the random integration of foreign DNA in the host genome following microinjection. Long genomic fragments are expected to contain all the regulatory elements necessary to induce an appropriate expression of transgenes. A bacterial artificial chromosome containing the porcine wap gene with approximately 145 and 5 kb of 5'- and 3'-flanking sequences, respectively, was microinjected into fertilized mouse ovocytes. In the six transgenic lines studied, expression was strictly specific to the mammary gland of lactating animals and was position-independent. Levels of exogenous porcine wap mRNA per copy compared favorably with the porcine wap mRNA yield in the mammary gland of a 9-day lactating pig. These findings suggest that this insert contained most if not all of the cis-acting elements involved in the full specific expression of the porcine wap gene. These elements constitute good candidates for directing the optimized expression of protein recombinant-encoding genes in the mammary gland of lactating animals. 相似文献
18.
Udo Kiessling Klaus Becker Michael Strauss Joerg Schoeneich Erhard Geissler 《Molecular & general genetics : MGG》1986,204(2):328-333
Summary This communication demonstrates the usefulness of the plamid rescue procedure for recovery of plasmids from transgenic mice. We have microinjected the plasmid pSK1 harbouring the Herpes simplex virus thymidine kinase gene into fertilized mouse oocytes and succeeded in recovering plasmids from newborns by transformation of E. coli either with HindIII cut cellular DNA or with uncut DNA. The majority of the rescued plasmids were indistinguishable from pSK1 by restriction analysis. The rescued plasmids proved to be functionally active in a transient expression assay in mouse Ltk- cells. The pSK1 DNA sequences were inherited by up to 90% of the second generation progeny mice, which is not in agreement with a Mendelian transmission of heterozygous markers integrated into a single site of the chromosome.These data support the assumption that germ line transmission of non-integrated episomal plasmids can occur. 相似文献
19.
Jin XL Guo H Mao C Atkins N Wang H Avasthi PP Tu YT Li Y 《Biochemical and biophysical research communications》2000,270(3):978-982
Emx1 is a mouse homologue of the Drosophila homeobox gene empty spiracles. Its expression is limited to the neurons in developing and adult cerebral cortex and hippocampus. Because of the highly restricted expression pattern of the Emx1 gene, it would be quite desirable to characterize the promoter of the Emx1 for directing foreign gene expression in the transgenic mouse. We report here that we have achieved the Emx1-specific expression in transgenic mice by inserting the lacZ reporter and cre genes directly into the exon 1 of the Emx1 gene using embryonic stem (ES) cell technology. The distribution of the beta-galactosidase activity in the transgenic mice was consistent with the published results obtained using in situ hybridization and immunohistochemistry. Furthermore, we have demonstrated that Cre protein was present in the cerebral cortex of the transgenic mice and was able to mediate loxP-specific recombination in vitro. The creation of this line of cre transgenic mice, and the demonstration that the insertion site located in the exon 1 of the Emx1 gene could render foreign genes a specific expression pattern restricted to the developing and adult cerebral cortex and hippocampus, should be conducive to further studies of the effect of a gene mutation or overexpression upon the development and plasticity of cerebral cortex and hippocampus. 相似文献
20.
Sperm cells as vectors for introducing foreign DNA into eggs: genetic transformation of mice 总被引:49,自引:0,他引:49
Mature mouse sperm cells incubated in an isotonic buffer with cloned DNA capture DNA molecules over a 15 min period. Spermatozoa incubated with pSV2CAT plasmid in either circular or linear form were used to fertilize mouse eggs in vitro. Sequences complementary to pSV2CAT were identified in approximately 30% of 250 progeny by Southern blotting. A genomic library was constructed from the DNA of a positive mouse. Three positive clones were identified and two adjacent HincII restriction fragments of 240 and 370 bp showed identical sequences to the corresponding fragments of the pSV2CAT plasmid. F1 progeny showed paternal and maternal transmission of the transgenes from founders. CAT gene expression was detected on tissues of adult F1 individuals, preferentially on tails and muscle. We conclude that transgenic mice can be obtained using sperm cells as foreign DNA vectors. 相似文献