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Genetic lineage tracing and conditional mutagenesis are developmental genetics techniques reliant on precise tissue-specific expression of transgenes. In the mouse, high specificity is usually achieved by inserting the transgene into the locus of interest through homologous recombination in embryonic stem cells. In the zebrafish, DNA containing the transgenic construct is randomly integrated into the genome, usually through transposon-mediated transgenesis. Expression of such transgenes is affected by regulatory features surrounding the integration site from general accessibility of chromatin to tissue-specific enhancers. We tested if the 1.2 kb cHS4 insulators derived from the chicken β-globin locus can shield a transgene from chromosomal position effects in the zebrafish genome. As our test promoters, we used two different-length versions of the zebrafish nkx2.5. We found that flanking a transgenic construct by cHS4 insulation sequences leads to overall increase in the expression of nkx2.5:mRFP. However, we also observed a very high degree of variability of mRFP expression, indicating that cHS4 insulators fail to protect nkx2.5:mRFP from falling under the control of enhancers in the vicinity of integration site. 相似文献
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Ovarian teratomas are tumors that arise from female germ cells and are often a mixture of immature embryonal carcinoma cells
and mature embryonic cells. Tissues derived from all three primary embryonic lineages (ectoderm, mesoderm, and endoderm) are
typically found in the mature elements of a teratoma. In the case of the transgenic mouse line TG.KD, created with an imprinted
transgene construct, malignant ovarian teratomas of a mixed germ cell tumor morphology occur in 15–20% of hemizygous female
carriers of the transgene. The tumors frequently metastasize and can result in death of the mouse. Genetic analysis indicates
that the tumors are associated with the transgene's integration site. Inbred FVB/N and female mice of other transgenic lines,
also created in the inbred FVB/N strain with the same DNA construct as TG.KD, do not develop teratomas. In addition to teratomas,
the integration of the transgene on Chromosome (Chr) 8 is associated with a perinatal lethality in homozygous transgenic carriers.
The hemizygous genotypes of the teratomas suggest that they arise from early germ cells, prior to the completion of meiosis
I.
Received: 31 August 1995 / Accepted: 3 November 1995 相似文献
4.
The random germline integration of genetically engineered transgenes has been a powerful technique to study the role of particular genes in variety of biological processes. Although the identification of the transgene insertion site is often not essential for functional analysis of the transgene, identifying the site can have practical benefit. Enabling one to distinguish between animals that are homozygous or hemizygous for the transgene locus could facilitate breeding strategies to produce animals with a large number of genetic markers. Furthermore, founder lines generated with the same transgene construct may exhibit different phenotypes and levels of transgene expression depending on the site of integration. The goal of this report was to develop a rapid protocol for the identification and verification of transgene insertion sites. To identify host genomic sequences at the coagulation Factor X transgene integration site, DNA from a tail snip of the transgenic mouse was digested with NcoI and circularized using T4 DNA ligase. Using appropriately positioned PCR primers annealing to a transgene fragment distal to a terminal transgene restriction site (NcoI), one could amplify a fragment containing the transgene terminal region and extending into the flanking genomic sequence at the insertion site. DNA sequence determination of the amplicon permitted identification of the insertion site using a BLASTN search. FISH analysis of a metaphase spread of primary fibroblasts derived from the transgenic mouse was consistent with the identification of insertion site near the end of mouse chromosome 14. Identification of transgene insertion sites will facilitate genotyping strategies useful for the construction of mice with multiple engineered genetic markers and to distinguish among different founder lines generated by the same transgene. Furthermore, identification of the insertion site is necessary to analyze unexpected phenotypes that might be caused by insertional inactivation of an endogenous gene. 相似文献
5.
High-resolution structural analysis of biolistic transgene integration into the genome of wheat 总被引:7,自引:0,他引:7
S. A. Jackson P. Zhang W. P. Chen R. L. Phillips B. Friebe S. Muthukrishnan B. S. Gill 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2001,103(1):56-62
Transformation of plant genomes by biolistic methods has become routine over the past decade. However, relatively little is
known about how transgenes are physically integrated into the host genome. Using a high-resolution physical mapping technique,
fluorescence in situ hybridization on extended DNA fibers (fiber-FISH), 13 independent transgenic wheat lines were analyzed
to determine the structural arrangement of stably inherited transgenes in host-plant chromosomes. Twelve transgenic lines
were transformed with a single plasmid and one line was co-transformed with two separate plasmids, which co-segregated genetically.
Three basic integration patterns were observed from the fiber-FISH experiments: Type I, large tandemly repeated integration;
Type II, large tandem integrations interspersed with unknown DNA; and Type III, small insertions, possibly interspersed with
unknown DNA. Metaphase FISH showed that the integration of transgenes was in both hetero- and euchromatic, as well as proximal,
interstitial and distal, regions of the chromosomes. In the transgenic plants, the type of promotor used, rather than the
chromosomal site of transgene integration, was most critical for transgene expression. The integration of the transgenes was
not associated with detectable chromosomal rearrangements.
Received: 25 August 2000 / Accepted: 31 October 2000 相似文献
6.
外源性人TIMP-1基因在转基因小鼠染色体上的整合及定位 总被引:1,自引:0,他引:1
为探讨外源基因人基质金属蛋白酶组织抑制物-1(human tissue inhibitor of metalloproteinase-1, hTIMP-1)基因在转基因小鼠家系染色体上的整合和精确定位,应用Southrn印迹检测外源基因在染色体上整合的位点及拷贝数.结果表明,外源基因是以单拷贝、单位点形式整合;应用荧光原位杂交(fluorescence in situ hybridization, FISH)技术检测F4~F20代转基因小鼠中外源基因的整合.结果证明,该家系转基因小鼠自F4代起是纯合子,外源基因整合在17号染色体E区;反向PCR法(Inverse PCR, IPCR)克隆出约3.8 kb外源基因整合位点处的侧翼序列.分析表明,外源基因整合在17号染色体E1.3区,ALK(anaplastic lymphoma kinase, ALK)基因第23个内含子区域.结果提示,获得的转基因小鼠为纯系,外源基因hTIMP-1已稳定整合在转基因小鼠染色体上,并能遗传给后代. 相似文献
7.
Plant transformation based on random integration of foreign DNA often generates complex integration structures. Precision in the integration process is necessary to ensure the formation of full-length, single-copy integration. Site-specific recombination systems are versatile tools for precise genomic manipulations such as DNA excision, inversion or integration. The yeast FLP-FRT recombination system has been widely used for DNA excision in higher plants. Here, we report the use of FLP-FRT system for efficient targeting of foreign gene into the engineered genomic site in rice. The transgene vector containing a pair of directly oriented FRT sites was introduced by particle bombardment into the cells containing the target locus. FLP activity generated by the co-bombarded FLP gene efficiently separated the transgene construct from the vector-backbone and integrated the backbone-free construct into the target site. Strong FLP activity, derived from the enhanced FLP protein, FLPe, was important for the successful site-specific integration (SSI). The majority of the transgenic events contained a precise integration and expressed the transgene. Interestingly, each transgenic event lacked the co-bombarded FLPe gene, suggesting reversion of the integration structure in the presence of the constitutive FLPe expression. Progeny of the precise transgenic lines inherited the stable SSI locus and expressed the transgene. This work demonstrates the application of FLP-FRT system for site-specific gene integration in plants using rice as a model. 相似文献
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Screening of animals to detect the presence of integrated DNA sequences is an essential component of transgenic mouse generation. Rapid and sensitive detection techniques to facilitate identification of transgenic animals for biological studies or subsequent breeding programs are desirable. Most transgenics are generated on F1 backgrounds, thus determination of the histocompatibility status of neonates provides important diagnostic information for establishing congenic colonies. We describe the application of two assays, in vitro DNA amplification using the polymerase chain reaction (PCR) and fluorescence in situ hybridization with biotinylated DNA probes, to facilitate rapid detection of transgenes and their chromosomal integration patterns in young mice. A noninvasive PCR-based assay to detect the transgene in DNA contained in detergent-extracted hair follicles was developed for rapid screening. A total of 147 mice derived from F2, F3, and F4 generations of C57BL x F1 (globin transgenics) were assayed to determine whether they carried a globin transgene. Characterization of animals by PCR-based amplification of the transgene was compared with that obtained using standard Southern analysis of DNA extracted from tails. Categorization of animals as positive (carrying the transgene) or negative using PCR was performed successfully in the initial assay with 95% of the animals. Fluorescence in situ hybridization with a DNA probe showing homology with a portion of the transgene was performed on metaphase and interphase cells to determine the integration pattern of the transgene. Our data showed that the transgene was integrated in a single chromosome. These techniques should facilitate rapid identification of transgenic animals and characterization of the genomic transgene integration patterns. 相似文献
11.
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. 相似文献
12.
Nancy Larochelle Rolf Stucka Norman Rieger Lothar Schermelleh Gudrun Schiedner Stefan Kochanek Eckhard Wolf Hanns Lochmüller 《Transgenic research》2011,20(1):123-135
Adenoviral vectors (AdV) are popular tools to deliver foreign genes into a wide range of cells. They have also been used in
clinical gene therapy trials. Studies on AdV-mediated gene transfer to mammalian oocytes and transmission through the germ
line have been reported controversially. In the present study we investigated whether AdV sequences integrate into the mouse
genome by microinjecting AdV into the perivitelline space of fertilized oocytes. We applied a newly developed PCR technique
(HiLo-PCR) for identification of chromosomal junctions next to the integrated AdV. We demonstrate that mouse oocytes can be
transduced by different recombinant adenoviral vectors (first generation and gutless). In one transgenic mouse line using
the first generation adenoviral vector, the genome has integrated into a highly repetitive cluster located on the Y chromosome.
While the transgene (GFP) was expressed in early embryos, no expression was detected in adult transgenic mice. The use of
gutless AdV resulted in expression of the transgene, albeit the vector was not transmitted to progeny. These results indicate
that under optimized conditions fertilized mouse oocytes are transduced by AdV and give rise to transgenic founder animals.
Therefore, adequate precautions should be taken in gene therapy protocols of reproductive patients since transduction of oocytes
or early embryos and subsequent chromosomal integration cannot be ruled out entirely. 相似文献
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Association of transgene integration sites with chromosome rearrangements in hexaploid oat 总被引:9,自引:0,他引:9
S. Svitashev E. Ananiev W. P. Pawlowski D. A. Somers 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2000,100(6):872-880
Transgene loci in 16 transgenic oat (Avena sativa L.) lines produced by microprojectile bombardment were characterized using phenotypic and genotypic segregation, Southern
blot analysis, and fluorescence in situ hybridization (FISH). Twenty-five transgene loci were detected; 8 lines exhibited
single transgene loci and 8 lines had 2 or 3 loci. Double FISH of the transgene and oat C- and A/D-genome-specific dispersed
and clustered repeats showed no preferences in the distribution of transgene loci among the highly heterochromatic C genome
and the A/D genomes of hexaploid oat, nor among chromosomes within the genomes. Transgene integration sites were detected
at different locations along individual chromosomes, although the majority of transformants had transgenes integrated into
subtelomeric and telomeric regions. Transgene integration sites exhibited different levels of structural complexity, ranging
from simple integration structures of two apparently contiguous transgene copies to tightly linked clusters of multiple copies
of transgenes interspersed with oat DNA. The size of the genomic interspersions observed in these transgene clusters was estimated
from FISH results on prometaphase chromosomes to be megabases long, indicating that some transgene loci were significantly
larger than previously determined by Southern blot analysis. Overall, 6 of the 25 transgene loci were associated with rearranged
chromosomes. These results suggest that particle bombardment-mediated transgene integration may result from and cause chromosomal
breakage and rearrangements.
Received: 29 July 1999 / Accepted: 9 November 1999 相似文献
15.
David Cunningham Qiang Xiao Aurobindo Chatterjee Kathleen Sulik Diana Juriloff Frederick Elder Wilbur Harrison Gabriele Schuster Paul A. Overbeek Gail E. Herman 《Mammalian genome》2002,13(4):179-185
Formation of the neural tube plays a primary role in establishing the body plan of the vertebrate embryo. Here we describe
the phenotype and physical mapping of a highly penetrant X-linked male-lethal murine mutation, exma (exencephaly, microphthalmia/anophthalmia), that specifically disrupts development of the rostral neural tube and eye. The mutation arose from the random
insertion of a transgene into the mouse X Chromosome (Chr). Eighty-three percent of transgenic male embryos display an open,
disorganized forebrain and lack optic vesicles. No transgenic males survive beyond birth. Hemizygous females show a variable
phenotype, including reduced viability and occasional exencephaly and/or microphthalmia. Altered or reduced expression patterns
of Otx2, Pax6, Six3, and Mrx, known markers of early forebrain and eye development, confirmed the highly disorganized structure of the forebrain and lack
of eye development in affected exma male embryos. Physical mapping of the transgene by FISH localized a single insertion site to the interval between Dmd and Zfx on the X Chr. A 1-Mb contig of BAC clones was assembled by using sequences flanking the transgene and revealed that the insertion
lies close to Pola1 and Arx, a gene encoding a highly conserved homeobox protein known to be expressed in the developing forebrain of the mouse. Data
from Southern blots of normal and transgenic DNA demonstrated that a large segment of DNA encompassing Arx and including part of Pola1 was duplicated as a result of the transgene insertion. From the physical mapping results, we propose a model of the gross
rearrangements that accompanied transgene integration and discuss its implications for evaluating candidate genes for exma. 相似文献
16.
Suchkova IO Baranova TV Kustova ME Kisljakova TV Vassiliev VB Slominskaja NO Alenina NV Patkin EL 《Tsitologiia》2004,46(1):53-61
Embryonal teratocarcinoma F9 cells were transfected with a fragment (3.8 kb) of bovine satellite DNA IV (Sat), which is not homologous to mouse satellite DNA. FISH analysis revealed various chromosomal integration sites of integrated Sat in different transsatellite clones. After several passages, transsatellite had a tendency to spread along chromosome bearing Sat in one of the studied lines. The integrated transsatellites were enriched with prolonged single-strand DNA regions (SSR) revealed by FISH without previous chromosomal denaturation, and were unmethylated. The observed SSR are presumably supposed to represent intermediates of transsatellite DNA instability via unequal sister chromatid exchanges. DAPI staining demonstrated that the integrated Sat induced the formation of prominent ectopic neoheterochromatin blocks in regions adjacent to integrated Sat. These blocks were located exclusively between integrated Sat and centromeric heterochromatin. Thus, mouse repetitive centromeric DNA (AT-rich, DAPI-positive) "spreads" along the chromosome in response to integration of the bovine satellite GC-rich DNA. The results obtained are discussed in the context of possible position effect variegation mechanisms operating in undifferentiated cells. 相似文献
17.
Shoichi Matsukuma Masaaki Kondo Mitsuyo Yoshihara Miyuki Matsuda Tadashi Utakoji Shizuyo Sutou 《Mammalian genome》1999,10(1):1-5
A line of transgenic mouse T604 transmitted a transgene to progeny together with a set of chromosomes with a reciprocal translocation.
The transgene was integrated at a single site in the translocated chromosomes, as revealed by fluorescence in situ hybridization.
The transgenic hemizygous males, also heterozygous for the translocation of chromosomes, showed apparently normal spermatogenesis,
while the males homozygous for the transgene as well as for the translocated chromosomes showed a defect in spermatogenesis.
Considering that the genetic rearrangement by either insertion of the transgene or the chromosome translocation in the T604
mouse line might have caused a recessive mutation in a gene indispensable for spermatogenesis, we have mapped the transgene
integration site and the translocation breakpoints in mouse chromosomes. Linkage analysis with SSLP markers showed that the
loci for the transgene and the translocation breakpoints were closely located to D5Mit24 on Chromosome (Chr) 5, and to a region between D19Mit19 and D19Jpk2 on Chr 19. Mea2 gene, mapped only 2 cM from D5Mit24 and known to show male-specific enhanced expression in the testis, was analyzed as a candidate for the gene disrupted in
T604 transgenic mice. Southern blot analysis revealed that Mea2 gene was indeed disrupted in T604 mice, and Northern blot analysis of the testis RNA showed that the expression of Mea2 was annihilated in the testis of T604 transgenic homozygotes.
Received: 8 July 1998 / Accepted: 23 September 1998 相似文献
18.
Charles Q. Cai Yannick Doyon W. Michael Ainley Jeffrey C. Miller Russell C. DeKelver Erica A. Moehle Jeremy M. Rock Ya-Li Lee Robbi Garrison Lisa Schulenberg Ryan Blue Andrew Worden Lisa Baker Farhoud Faraji Lei Zhang Michael C. Holmes Edward J. Rebar Trevor N. Collingwood Beth Rubin-Wilson Philip D. Gregory Fyodor D. Urnov Joseph F. Petolino 《Plant molecular biology》2009,69(6):699-709
Targeted transgene integration in plants remains a significant technical challenge for both basic and applied research. Here
it is reported that designed zinc finger nucleases (ZFNs) can drive site-directed DNA integration into transgenic and native
gene loci. A dimer of designed 4-finger ZFNs enabled intra-chromosomal reconstitution of a disabled gfp reporter gene and site-specific transgene integration into chromosomal reporter loci following co-transformation of tobacco
cell cultures with a donor construct comprised of sequences necessary to complement a non-functional pat herbicide resistance gene. In addition, a yeast-based assay was used to identify ZFNs capable of cleaving a native endochitinase
gene. Agrobacterium delivery of a Ti plasmid harboring both the ZFNs and a donor DNA construct comprising a pat herbicide resistance gene cassette flanked by short stretches of homology to the endochitinase locus yielded up to 10% targeted,
homology-directed transgene integration precisely into the ZFN cleavage site. Given that ZFNs can be designed to recognize
a wide range of target sequences, these data point toward a novel approach for targeted gene addition, replacement and trait
stacking in plants. 相似文献
19.
Structural features of the integration site of foreign DNA in the transgenic mouse genome 总被引:2,自引:0,他引:2
The structure of the transgenic mouse DNA region containing an integrated transgene (fragment of pBR322 sequence) was analysed. In one of the sequences flanking the transgene, short direct and inverted overlapping repeats were revealed at a distance of 60 bp from the integration site. In the same flanking sequence, there is an extended sequence (3.5 kbp) 0.3-1 kbp away from the transgene. It repeats 100-300 times in the mouse genome and is highly conservative (the homologs of the repeat have been revealed in other mammalian, bird, fish and insect genomes). This up-to-date unknown family of highly-conserved dispersed repeats has been denoted by T1. We believe that both the revealed short inverted repeats capable of forming hairpins with loops and the T1 repeat are structures involved in the process of non-homologous insertion of foreign DNA into the region of the transgenic mouse genome. 相似文献
20.
Popov AV Smirnov AF Suchkova IO Baranova TV Sorokin AV Gaĭtskhoki VS Patkin EL 《Genetika》2000,36(8):1119-1125
Transgenic mice carrying bovine satellite DNA IV were obtained. The size of the transgene integrated into the mouse genome was approximately 390 kb (about 100 transgene copies) as determined by a semiquantitative PCR. Restriction analysis with isoschizomeric restrictases HpaII and MspI, showed that the alien DNA was methylated. In the genome of a transgenic founder male, two integration sites for satellite DNA IV were revealed by in situ hybridization and in situ PCR. These sites are situated on two different chromosomes: in pericentromeric heterochromatin and within a chromosomal arm. In transgenic mice, de novo formation of heterochromatin regions (C-block and the CMA3 disk within the centromeric heterochromatin of another chromosome) was revealed by C-banding and staining with chromomycin A3. This formation is not characteristic of mice, because their chromosomes normally contain no interstitial C-blocks or sequences intensely stained by chromomycin A3. 相似文献