Detection of Y-bearing porcine spermatozoa by in situ hybridization using digoxigenin-labeled,porcine male-specific DNA probe produced by polymerase chain reaction

This study was carried out to determine whether Y-bearing porcine spermatozoa could be detected by in situ hybridization using a digoxigenin (Dig)-labelled DNA probe specific to the Y chromosome produced by polymerase chain reaction (PCR). A conventional PCR (with Dig-dUTP) was performed using a set of oligonucleotide primers (5′-AAGTGGTCAGCGTGTCCATA-3′ and 5′-TTTCTCCTGTATCCTCCTGC-3′) for 236 bp fragment of porcine male-specific DNA sequence and 1.25 × 10⁴ template white blood cells obtained from a boar. When fluorescence in situ hybridization with the Dig-labelled DNA probe was applied to the metaphase chromosome spreads prepared from both boar and gilts, the fluorescein signal was only detected on the long arm of the Y chromosome. In addition, immunocytochemical detection with the Dig-labelled DNA probe and alkaline phosphatase-labeled anti-Dig was applied to both sperm nuclei pretreated with dithiothreitol and white blood cells; 51% of sperm nuclei and 96% of white blood cells obtained from boar were labelled, whereas none of white blood cells obtained from gilts were labelled with the Dig-labelled DNA probe. The results indicated that in situ hybridization with porcine male-specific DNA probe produced by PCR made possible the direct visualization of Y-bearing porcine spermatozoa by in situ hybridization. © 1995 Wiley-Liss, Inc.     

Sex-related growth rate differences in mouse preimplantation embryos in vivo and in vitro

Sex-related growth rate differences in preimplantation mouse embryos were investigated. In experiment I, Day 3 embryos were recovered from reproductive tracts, classified according to developmental stage, and cultured for 24 hr in CZB medium containing glucose. Each embryo was then reclassified and stained for measurement of number of nuclei and finally sexed using the polymerase chain reaction. In experiment II, Day 4 embryos were recovered, classified, stained, and sexed as in experiment I immediately after recovery. Morphologically, there were no differences between the sexes in either of the experiments on Day 4. However, based on number of nuclei, the data showed that in vitro conditions support the development of male embryos to the blastocyst stage compared to female embryos. Furthermore, growth rate differences were observed in vivo on Day 3, as females compacted earlier than males. These results suggest that the increased cell proliferation in cultured male embryos is an artifact caused by the in vitro environment. The variation may be due to sex differences in embryonal energy metabolism during the preimplantation stage. The growth difference implies different in vitro requirements of male and female embryos. © 1995 Wiley-Liss, Inc.     

Evaluation of mass spectrometric techniques for charaterization of engineered proteins

A simple and versatile method of in vitro site-specific mutagenesis based on polymerase chain reaction (PCR) is described. The complete method required the use of three oligonucleotide primers and two PCRs. The product of the first PCR was used as one of the primers (megaprimer) in the second PCR. Essentially 100% of the final product incorporated the desired mutation. The various aspects of the procedure and its application is described in detail.     

The biotechnology and applications of antibody engineering

The exquisite specificity of monoclonal antibodies (MAb) has long provided the potential for creating new reagents for the in vivo delivery of therapeutic drugs or toxins to defined cellular target sites or improved methods of diagnosis. However, many difficulties associated with their production, affinity, specificity, and use in vivo have largely confined their application to research or in vitro diagnostics. This situation is beginning to change with the recent developments in the applied molecular techniques that allow the engineering of the genes that encode antibodies rather than the manipulation of the intact antibodies themselves. Techniques, such as the polymerase chain reaction, have provided essential methods with which to generate and modify the genetic constituents of antibodies, allow their conjugation to toxins or drugs, provide ways of humanizing murine antibodies, and allow discrete modular antigen binding components to be produced. More recent developments of in vitro expression systems and powerful phage surface display technologies will without doubt play a major role in future antibody engineering and in the successful development of new diagnostic and therapeutic antibody-based reagents.     

Specific monitoring by PCR amplification and bioluminescence of firefly luciferase gene-tagged bacteria added to environmental samples

Abstract The firefly luciferase gene, luc , was demonstrated to hold promise as a specific marker for monitoring of genetically modified bacteria in the environment. PCR amplification and bioluminescence procedures were modified and compared for environmental monitoring of luc -tagged bacteria, using Escherichia coli as a model. The methods were used to track luc -tagged bacterial cells added to intact sediment core microcosms. Detection limits for the luc -tagged cells were the following, expressed as cells per 0.5 g of sediment: 10², by PCR amplification; 10³, by whole cell luminescence; and 10³−10⁴, by measurement of luminescence in cell extracts.     

Purification and PCR-based cDNA cloning of a plastidial n-6 desaturase

A plastidial membrane-bound n-6 desaturase from spinach (Spinacia oleracea) was purified from chloroplast envelope membranes by anion exchange, cation exchange and ferredoxin-affinity chromatography. The molecular mass of the protein was estimated by SDS-PAGE to be 40 kDa. The highest specific activity of the desaturase in the final preparation was 196 nmol/min per mg protein with free oleic acid as the substrate. The N-terminal amino acid sequence of the blotted protein was determined and used for the construction of a degenerated and inosine-containing oligonucleotide primer for PCR experiments with cDNA transcribed from leaf mRNA. A 3-RACE experiment with this primer amplified a single band of 1500 bp that after sequencing showed an open reading frame of 382 amino acids corresponding to a protein of 43 kDa. The 5 end of the cDNA was amplified by a 5-RACE experiment and isolated as a 500 bp fragment. Sequencing of this DNA revealed an additional 65 amino acids at the N-terminus of the native protein that are attributed to a plastidial leader peptide. With appropriate primers derived from these sequences a full-length clone was amplified by PCR and sequenced. Comparison of the plastidial oleate desaturase with the homologous enzyme from cyanobacteria showed about 50% amino acid homology. Comparison with other desaturases revealed three histidine boxes with the general sequence HXXXH that are highly conserved in all membrane-bound desaturases. These boxes might be involved in metal ion complexation required for reduction of oxygen.     

旋毛虫肌幼虫ES抗原的基因克隆及高效表达

作者对编码旋毛虫肌幼虫ES抗原的部分结构基因进行了克隆、鉴定和表达。用RNA PCR技术直接从旋毛虫肌幼虫总RNA中反转录并扩增出0．7kh的靶DNA,酶切分析后将其克隆到融合表达载体pEx3lC中。SDS—PAGE电泳表明,含重组子的大肠杆菌能够表达出一分子量为37kDa的融合蛋白(P37),后者占菌体总蛋白的22%以上,并以包含体形式存在于菌体中。经对纯化后表达蛋白的ELlSA检测,证明它能被猪旋毛虫病阳性血清和抗旋毛虫单克隆抗体识别。研究结果揭示,重组蛋白P37对于研制旋毛虫病诊断抗原和免疫抗原具有潜在的应用价值。