We have transferred a gene coding for rabbit (3-globin into the male pronucleus of mouse zygotes by direct microinjection. Some of these zygotes developed into mature mice which contained this gene and appeared to be producing a rabbit globin. Evidence for the presence ofthe gene in these animals was provided by Southern blot hybridization analysis. Evidence for the expression of the rabbit gene in these transformed mice and their offspring was provided by hemoglobin isoelectric focusing analysis and specific serological reactivity between mouse anti-rabbit hemoglobin antiserum and a hemolysate from the mice that developed from the microinjected zygotes. The use of this zygote transformation may allow the introduction and expression of a broad range of genetic elements in mammals. Fertilization is a complex, multistep phenomenon initiated by the interaction and fusion of the spermatozoon and egg and culminated by the association of the two groups of chromosomes, one derived from the maternal pronucleus and the other derived from the paternal pronucleus. During the early stages of the fertilization process, just after the spermatozoon penetrates into the egg, the sperm nucleus undergoes a complex chromosomal decondensation process (11-13) after which the male chromosomal complement appears to undergo changes in chromosomal composition prior to merging with the female pronucleus (14). Although little is known about this stage of fertilization in the mammal, studies of the sea urchin suggest that significant changes within the male pronucleus and chromatin are brought about by proteins emanating from the oocyte cytoplasm and female pronucleus (15, 16). Microinjection of specific DNA sequences into the male pronucleus during pronuclear "processing" ofthe male chromosomes might result in the appropriate delivery ofthese sequences into the zygote nucleus along with the male chromosomal complement.On the basis of these considerations, an estimated 20,000 copies ofeither purified rabbit (globin gene fragment or a rabbit (3-globin gene-containing plasmid were microinjected into male pronuclei of mouse zygotes. The resulting embryos were cultured in vitro to morulae or blastocysts and transferred into pseudopregnant foster mothers. We report here not only the presence of rabbit (3-globin gene in offspring developed from these microinjected mouse zygotes but also serological and electrophoretic evidence for the production of a rabbit globin protein in these mice.MATERIALS AND METHODS Collection ofZygotes. Fertilized.eggs at the pronuclear stage (the male and female pronuclei being separated and distinguishable within the cytoplasm) were collected from the oviducts of C57BL/6J females that had been mated to LT/Sv males. After removal of the surrounding cumulus cells in culture medium (17) with bovine testis hyaluronidase (1 mg/ml), pooled zygotes from several females were washed in fresh medium and stored, until micromanipulation, in a depression slide containing culture medium overlayered with paraffin oil in an a...
The severe acute respiratory syndrome coronavirus (SARS-CoV) is the causative agent of the recent outbreak of severe acute respiratory syndrome. VeroE6 cells, fetal rhesus monkey kidney cells, and human peripheral blood mononuclear cells were the only cells known to be susceptible to SARS-CoV. We developed a multiplex reverse transcription-PCR assay to analyze the susceptibility of cells derived from a variety of tissues and species to SARS-CoV. Additionally, productive infection was determined by titration of cellular supernatants. Cells derived from three species of monkey were susceptible to SARS-CoV. However, the levels of SARS-CoV produced differed by 4 log 10 . Mink lung epithelial cells (Mv1Lu) and R-Mix, a mixed monolayer of human lung-derived cells (A549) and mink lung-derived cells (Mv1Lu), are used by diagnostic laboratories to detect respiratory viruses (e.g., influenza virus); they were also infected with SARS-CoV, indicating that the practices of diagnostic laboratories should be examined to ensure appropriate biosafety precautions. Mv1Lu cells produce little SARS-CoV compared to that produced by VeroE6 cells, which indicates that they are a safer alternative for SARS-CoV diagnostics. Evaluation of cells permissive to other coronaviruses indicated that these cell types are not infected by SARS-CoV, providing additional evidence that SARS-CoV binds an alternative receptor. Analysis of human cells derived from lung, kidney, liver, and intestine led to the discovery that human cell lines were productively infected by SARS-CoV. This study identifies new cell lines that may be used for SARS-CoV diagnostics and/or basic research. Our data and other in vivo studies indicate that SARS-CoV has a wide host range, suggesting that the cellular receptor(s) utilized by SARS-CoV is highly conserved and is expressed by a variety of tissues.
A simplified DNA hybridization method was developed to detect acyclovir-resistant isolates of herpes simplex virus. Herpes simplex virus-infected cell cultures in microtiter plates were treated with concentrations of acyclovir ranging from 8 to 0.015 ,ug/ml. At 48 h postinfection, infected cells were lysed by a one-step procedure and lysates were absorbed to membranes. Without further treatment, membranes were hybridized by using a herpes simplex virus-specific radioiodinated probe. The membranes were then washed and counted in a gamma counter. The elapsed time for assay performance was 4 h. Parallel plaque reduction assays were performed for comparison. The mean 50% inhibitory dose of in vivo-and in vitro-derived acyclovir-resistant, thymidine kinase-negative isolates was greater than 2 ,ug/ml by DNA hybridization. The 50% inhibitory dose of acyclovir-susceptible, thymidine kinase-positive isolates ranged from 0.01 to 1.1 ,ug/ml. This assay is simple and objective and should facilitate antiviral susceptibility testing in diagnostic laboratories.Acyclovir (ACV), a purine nucleoside, is used for treatment and suppression of infections due to herpes simplex virus (HSV;4,9,15,23,24). The drug is phosphorylated by viral thymidine kinase (TK) to acyclovir monophosphate and then phosphorylated by cellular enzymes to the triphosphate form (7, 16). The triphosphate form inhibits the viral DNA polymerase which ultimately results in termination of HSV DNA synthesis (6, 12). Clinical resistance is encountered infrequently and is generally associated with mutations in the TK gene (11). However, with recent Food and Drug Administration approval of oral ACV, widespread use of the drug is likely to develop and may create a selective environment favoring the emergence of resistance. Greater use of ACV may stimulate increased interest in screening HSV isolates for ACV resistance.Gadler et al. (8) successfully employed a conventional DNA hybridization procedure which measured the effects of antiviral compounds on HSV replication. The procedure these investigators described used 32p, required many tedious manipulations, and is not practical for routine use. The present study describes the application of a recently developed, simplified DNA hybridization procedure to detect acyclovir-resistant (ACV9 HSV isolates and compares the sensitivity of this new procedure with that of the present reference method of plaque reduction. This hybridization assay is applicable by diagnostic laboratories and allows convenient testing of multiple antiviral agents.(
A rapid assay was developed to screen for herpes simplex virus (HSV) isolates that are resistant to acyclovir and other antiviral agents. The assay is a modified plaque reduction assay (PRA) in which the number of plaques seen in the absence of acyclovir was compared with that seen in the presence of a single cutoff concentration of acyclovir (2 microg/mL). This assay utilizes a cell line that expresses beta-galactosidase only after infection with HSV. Since histochemically stained plaques are easily visualized, small plaques can be easily enumerated. This allows the assay to be performed on dilutions of untitered specimens in the small wells of a 24-well plate and allows the results to be read only 2 days after inoculation of the virus. The assay performed well compared with a standard PRA and should be a valuable tool in identifying drug-resistant HSV in a timely manner.
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