Alan L. Goldin

Maintenance of Xenopus laevis and oocyte injection

Publisher Summary This chapter explains the maintenance of Xenopus laevis and oocyte injection. There are six species of Xenopus , all indigenous to Africa, but Xenopus laevis is the only one commonly used in laboratory research. To synchronize oocyte development, the frogs can be injected with human chorionic gonadotropin (hCG), which induces ovulation and egg laying, after which oocyte development starts over approximately synchronously. Xenopus can tolerate a wide range of temperature fluctuations, but the quality of oocytes diminishes markedly following temperature shifts. Therefore, frogs should be housed in a temperature-controlled environment. Xenopus are very susceptible to desiccation, and they can die from being out of the water for a few hours. Oocytes from Xenopus laevis can be classified into six stages of development on the basis of anatomy; the choice of which stage oocytes to inject depends on the purposes of the experiment.

High-frequency transfer of cloned herpes simplex virus type 1 sequences to mammalian cells by protoplast fusion.

The protoplast fusion technique of Schaffner (W. Schaffner, Proc. Natl. Acad. Sci. U.S.A. 77:2163-2167, 1980) has been adapted to introduce cloned herpes simplex virus genes into cultured mammalian cells. The technique involves digesting bacterial cell walls with lysozyme to produce protoplasts and then fusing the protoplasts to mammalian cells by treatment with polyethylene glycol. For monitoring transfer, protoplasts were labeled with the fluorescent dye fluorescein isothiocyanate before fusion. After fusion, greater than 50% of the mammalian cells were fluorescent, demonstrating that bacterial material was transferred with high frequency. Transfer of plasmid pBR325 occurred at frequencies of 1 to 2%, as measured by in situ hybridization. Fusion transfer of a chimeric plasmid consisting of the herpes simplex virus type 1 (strain KOS) EcoRI fragment F in pBR325 resulted in expression of some viral genomic sequences in about 5% of the mammalian cells, as detected by indirect immunofluorescence. One Ltk- cell in 300 to 500 was transformed to the TK+ phenotype after fusion with protoplasts carrying the chimeric plasmid pX1, which consists of pBR322 and the BamHI fragment coding for the herpes simplex virus type 1 thymidine kinase gene.

Expression of herpes simplex virus beta and gamma genes integrated in mammalian cells and their induction by an alpha gene product.

The proteins of herpes simplex virus type 1 (HSV-1) form three kinetic groups termed alpha, beta, and gamma, whose synthesis is regulated in a cascade fashion. alpha products are synthesized first during infection, and they are required for synthesis of beta and gamma proteins. To examine the expression of several HSV-1 beta and gamma genes in the absence of alpha functions, we transferred into mammalian cells a plasmid containing a region of the HSV-1 genome that codes for only beta and gamma genes (0.315 to 0.421 map units). We found stable integration of at least one copy of the intact plasmid in each cell line. Four HSV-1 transcripts of the beta and gamma classes were transcribed constitutively in the cells, including the genes for glycoprotein B and DNA-binding protein. No constitutive synthesis of these two proteins could be demonstrated, however. The integrated HSV-1 genes responded to viral regulatory signals in that they could be induced by infection with HSV-1 mutants resulting in a high level of synthesis of both glycoprotein B and DNA-binding protein. The HSV-1 alpha gene product ICP4 was necessary for this induction, and it was found to be most efficient at a low multiplicity of infection. Functional expression of four genes was demonstrated in that the cell lines complemented infecting HSV-1 temperature-sensitive mutants. The same genes were not available for homologous recombination with infecting virus, however, since no recombinant wild-type virus could be detected. These data demonstrate that HSV-1 beta and gamma genes can be transcribed in the absence of alpha functions in mammalian cells, but that they still respond to HSV-1 regulatory signals such as the alpha gene product ICP4.

[27] High-efficiency transfer of DNA into eukaryotic cells by protoplast fusion

Protoplast fusion is a highly efficient method for effecting gene transfer to cells in culture resulting in stable transformation at high frequency. A number of cell lines have been used successfully as recipients. There is no need to isolate and purify the DNA, which not only saves time and effort but eliminates steps that cause nicking or breaking of large cloned inserts. The high-frequency transformation achievable by protoplast fusion should make this procedure useful for studies on gene expression and for screening cloned genomic libraries for genes that can be expressed in recipient cells.

Herpes simplex virus thymidine kinase gene is stably maintained and expressed in cells transformed by protoplast fusion

We examined a series of transformed cell lines resulting from transfer of the herpes simplex virus type 1 thymidine kinase gene to Ltk− cells by protoplast fusion gene transfer. We show that multiple copies of the transforming plasmid DNA, ranging from a minimum of two to greater than 20, were present in one or at most a few integration sites in each cell line. The TK+ phenotype was stable in five independent transformed cell lines after growth in nonselective medium for over a year. Transforming plasmid DNA was stable in one cell line containing from two to five copies after a year of growth in nonselective medium. In another cell line initially containing about 20 copies, the transforming DNA became rearranged soon after growth to mass culture, resulting in a decrease to two to five copies which then remained stably maintained. This suggests that TK+ transformants resulting from protoplast fusion are stable when the input DNA has integrated in a relatively low copy number.

Stable induction of a 51K cellular protein in neuronal cells surviving herpes simplex virus type 1 infection

A series of survivor cell lines derived by infection of B103 rat neuroma cells with active wild-type herpes simplex virus type 1 (HSV-1) (M. Levine, A. L. Goldin, and J. C. Glorioso, J. Virol. 35, 203-210 (1980)) has been isolated. The survivor cells produced no infectious virus, yet they continued to react with HSV-1 antiserum for over 100 cell generations following the initial infection. The reactivity of the survivor cells with HSV-1 antiserum is characterized as being due to expression of a 51K protein. The 51K protein reacted with antiserum prepared against HSV-1 virions and was not detectable in the parental B103 cells. A protein of the same molecular weight was seen in productively infected B103 and HEL cells. The protein detected in the survivor cells comigrated with that seen in the infected cells on two-dimensional gel electrophoresis, indicating that they represent similar proteins. Despite the presence of the 51K protein reactive with HSV-1 antiserum, the survivor cells contain no detectable HSV-1 DNA sequences. They do contain DNA sequences which cross-hybridize with HSV-1 DNA, but similar cross-hybridizing sequences were also present in the parental B103 cells. No hybridizing polysomal, polyadenylated RNA species were present in the survivor cells that were not present in the parental B103 cells when probed with the cross-hybridizing HSV-1 restriction fragments. Therefore, the 51K protein evidently represents a cellular protein induced by the HSV-1 infection.