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101.
Masahiro Sato Norihiro Tada Reiko Iwase Egon Amann 《Molecular reproduction and development》1993,34(4):349-356
It is a well-known phenomenon that cultured mammalian cells that have been pricked in the presence of foreign DNA can be transformed. This micromanipulation ‘pricking’ technique was applied to mouse blastocysts to determine whether uptake of exogenous DNA would occur in the embryos. The middle region of the inner cell mass (ICM) was pricked three times in each blastocyst in a medium containing a linearized plasmid DNA. When the 60 treated blastocysts were transferred to the uterine horns of pseudopregnant females, 30 developing fetuses (50%) at the mid-gestation stage were obtained. Twenty-two of the 30 fetuses (73%) had less than 1 copy of the foreign DNA per diploid cell, as revealed by polymerase chain reaction (PCR)-Southern analysis, a sensitive technique combined with Southern blot processing of the PCR products. The 8 other fetuses were negative for the foreign DNA. When blastocysts were pricked in the presence of vector DNA coupling E. coli β-galactosidase (β-gal) gene to a mouse metallothionein-I (MT-I) promoter and assessed for β-gal activity histochemically after 1 and 5 days of culture in the presence of 1 μM CdCI2, at least 65% of the embryos exhibited β-gal activity mainly in the ICM region. These results indicate that mouse blastocysts can be transfected with a relatively high efficiency after pricking, and that the introduced gene expression occurs. This approach provides a means of mapping the regulatory elements of genes that are active in the mouse blastocyst ICM, and may be useful in investigating the fate of the ICM cells in an intact blastocyst by labeling them via pricking technique. © 1993 Wiley-Liss, Inc. 相似文献
102.
用DNA聚合酶链反应检测单纯疱疹病毒特异性核酸,调查100名孕产妇生殖器官脂HSV感染和羊水受HSV污染的情况,结果表明:受感染的母体通过病毒护散途径及通过产道途径,污染羊水的机率分别为11.1%和54.5%;原发性HSV、继发性HSV垂直污染羊水机率分别为66.7%和29.4%;不同妊娠期和生育胎次对污染率无显著性影响。 相似文献
103.
104.
K. B. C. Appa Rao C. H. Pawshe S. M. Totey 《Molecular reproduction and development》1993,36(3):291-296
This study was conducted to determine the sex of buffalo embryos produced in vitro by amplifying male specific DNA sequences using the polymerase chain reaction (PCR). This method uses three different pairs of bovine Y-chromosome specific primers and a pair of bovine satellite specific primers. Buffalo in vitro fertilized embryos at the 4-cell to blastocyst stage were collected at days 3, 4, 6, and 8 postinsemination, and the sex of each embryo was determined using all three different Y-chromosome specific primers. The bovine satellite sequence specific primers recognize similar sequences in buffalo and are amplified both in males and in females. Similarly, Y-chromosome specific primers amplify the similar Y-chromosome specific sequences in male embryos of buffalo. Upon examining genomic DNA from lymphocytes of adult males and females, and embryos, the results demonstrate the feasibility of embryo sexing in buffaloes. Furthermore, sex determination by PCR was found to be a rapid and accurate method. © 1993 Wiley-Liss, Inc. 相似文献
105.
Abstract Polymerase chain reaction was carried out to amplify the conserved region (789 bp in the case of Mycoplasma capricolum ) of the dnaA gene (1350 bp in the case of M. capricolum ) of 15 representatives of the class Mollicutes using degenerate oligonucleotide primers. The dnaA gene fragments were amplified from M. mycoides subsp. capri, Spiroplasma apis and S. citri . The amino acid sequences deduced from the nucleotide sequences of the amplified fragments showed very low similarities to those of the corresponding regions of four walled bacteria. The values of similarity between any two of the three mollicute species were lower than those between any two of the four walled bacteria. 相似文献
106.
Karen Valverde-Méndez Eduardo J. Hernández Dennis Matamoros Natalia Barboza 《The Annals of applied biology》2023,183(3):231-243
Begomoviruses (Geminiviridae family) are characterized by their high recombination rate and a wide range of hosts, making their control difficult. In Costa Rica, various species of bipartite begomoviruses have been reported, which are Pepper golden mosaic virus (PepGMV), Tomato yellow mottle virus (ToYMoV), Tomato leaf curl Sinaloa virus (ToLCSiV) and the monopartite begomovirus Tomato yellow leaf curl virus (TYLCV). Since the TYLCV first report in Costa Rica, neither additional knowledge has been produced on how this begomovirus has spread in the country's territory nor on the distribution of the other bipartite species. A total of 429 tomato samples collected during the years 2015–2016 were used to study these aspects. Each sample was georeferenced and analysed with various techniques such as nucleic acid hybridization, polymerase chain reaction (PCR) and sequencing for the begomoviruses previously reported in Costa Rica. It was found that the presence/absence of the different species can vary, depending on the province. TYLCV is present in the six provinces analysed in this work, with a proportion from 3.7 to 86.6 per cent. Alajuela, Cartago, and Heredia are the provinces most affected by tomato-infecting begomoviruses. Fourteen different haplotypes of TYLCV were detected, but all were identified as TYLCV-IL. The distribution of TYLCV was related to the presence of the whitefly Bemisia tabaci MED, especially in the country's main tomato production areas. This information allows the phytosanitary surveillance services to develop strategies for the integrated management of the disease and to contribute data to the genetic improvement programmes of the crop. 相似文献
107.
烟草耐盐愈伤组织变异体对盐渍的适应性 总被引:1,自引:0,他引:1
用组织培养及逐步增加,NaCl浓度的方法,筛选出烟草耐盐愈伤组织变异体。能耐盐(2% NaCl)的愈伤组织在2% NaCl中继代29次,再移入无盐培养基中培养11和20代后不能保持提高的耐盐性,分别退化到只耐1.5%与1.0% NaCl的水平。耐2% NaCl愈伤组织产生的再生植株自交后代,其萌发种子、幼苗及成长植株均未能表现出耐盐性,说明用选择胁迫方法所筛选出的耐盐细胞系,其耐盐性的提高属于生理适应性。 相似文献
108.
Rapid assessment of single-copy nuclear DNA variation in diverse species 总被引:12,自引:0,他引:12
We investigated the use of PCR primers designed to conserved exons within nuclear DNA to amplify potentially variable regions such as introns or hypervariable exons from a wide range of species. We then explored various approaches to assay population-level variation in these PCR products. Primers designed to amplify regions within the histone H2AF, myoglobin , MHC DQA , and aldolase (ALD) genes gave clean amplifications in diverse mammals (DQA) , and in birds, reptiles and mammals ( aldolase, H2AF, myoglobin ). The sequenced PCR products generally, but not always, confirmed that the correct locus had been amplified. Several primer sets produced smaller size fragments consistent with preferential amplification of intronless pseudogenes; this was confirmed by sequencing seal and reptile H2AF PCR products. Digestion with randomly selected four-base recognizing enzymes detected variation in some cases but not in others. In species/gene combinations with either low (e.g. seal H2AF, ALD-A ) or high (e.g. skink ALD-1 ) nucleotide diversity it was more efficient to sequence a small number of distantly related individuals (e.g. one per geographic population) and from these data to identify informative or potentially informative restriction enzymes for 'targeted' digestion. We conclude that for studies of population-level variation, the optimal approach is to use a battery of primers for initial PCR of both mtDNA and scnDNA loci, select those that give clean amplifications, and sequence one sample from each population to (i) confirm gene identity, (ii) estimate the amount of variation and, (iii) search for diagnostic restriction sites. This will allow determination of the most efficient approach for a large-scale study. 相似文献
109.
Cucumber mosaic virus genome is encapsidated in alfalfa mosaic virus coat protein expressed in transgenic tobacco plants 总被引:1,自引:0,他引:1
The expression of viral coat protein (CP) in transgenic plants has been shown to be very effective in virus plant protection. However, the introduction of CP genes into plants presents the potential risk of the encapsidation of a superinfecting viral genome in the transgenic protein, an event which could change the epidemiology of the disease. To detect the potential heterologous encapsidation of the cucumber mosaic virus (CMV) genome by alfalfa mosaic virus (AIMV) CP expressed in transgenic tobacco plants, a system of immunocapture (IC) and amplification by polymerase chain reaction (PCR) was optimized. This provided high sensitivity and reliable selection of the heterologously encapsidated CMV genome in the presence of natural CMV particles. As little as 2 pg of virus could be detected by immunocapture/polymerase chain reaction (IC/PCR) technique. Evidence for heterologous encapsidation of the CMV genome was found in 11 of the 33 transgenic plants tested two weeks after CMV inoculation. This demonstrates a significant rate of heterologous encapsidation events between two unrelated viruses in transgenic plants. Since CP is involved in the interactions of the virus particle with its vector, the release in the field of such transgenic plants could alter the transmission properties of some important viruses. 相似文献
110.
Wing Y. Cheung Jean-Charles Côté Diane L. Benoit Benoit S. Landry 《Plant Molecular Biology Reporter》1993,11(2):142-155
We have designed a simple and rapid assay for chloroplast-based triazine resistance in higher plants using PCR amplification
of thepsbA gene coupled toMaeI digestion of the amplified product to distinguish triazine resistant from sensitive biotypes. Our assay is universal and
avoids the need of lengthy procedures of previously published assays, which either required spraying of seedlings in a controlled
environment, quantification of chlorophyll fluorescence of leaf discs after incubation in triazine solution, DNA sequencing
of thepsbA gene, or Southern-blot analysis. Our diagnostic system is qualitative, reliable, fast and simple. More than 100 seedlings
taken directly from the field can be analyzed in one day. This system has a direct application towards a more rational use
of herbicides in production fields. It also represents a valuable tool to monitor spreading of resistant biotypes through
time and space and can serve as a model system applicable to other gene monitoring needs. 相似文献