Genetic Dissection of Neural Properties using Somatic Cell Hybrids

A set of neuronal genes can be expressed in neuroblastoma × L cell hybrids and hybrid cell lines with specific defects in neural function can be generated in high yield.     

Loss of Human Genetic Markers in Man-Chinese Hamster Somatic Cell Hybrids

MAN-MOUSE somatic cell hybrids are useful for the study of genetic linkage in man because the human chromosomes are preferentially lost and most murine and human forms of homologous isozymes are clearly distinguishable^1,2. There are, however, certain limitations in this system which call for the introduction of other interspecific somatic cell hybrids. (1) Not all the enzyme phenotypes of man and mouse are easily distinguishable by conventional electrophoretic procedures. (2) Groups of human chromosomes may be preferentially retained or lost in the man-mouse hybrids³. We do not know whether such groups form regular patterns and are constant for a particular hybrid type. (3) The frequency and types of chromosomal rearrangements, which has been reported in man-mouse hybrids, might be different in other human interspecific hybrids.     

Survival and DNA Repair of Somatic Cell Hybrids after Ultraviolet Irradiation

THE technique of somatic cell hybridization has opened up studies on genetic regulation¹ and human genetic analysis^2–5. Hybrid cells are isolated in conditions that select against parental cells while allowing hybrids to survive by genomic complementation. In xeroderma pigmentosum (XP), a human disease with an autosomal recessive defect in an early stage of DNA repair⁶, the skin is extremely sensitive to sunlight in vivo⁷ and skin fibroblasts show sharply reduced survival following ultraviolet irradiation in vitro^8,9. This communication concerns the use of ultraviolet irradiation in combination with a chemical method to produce hybrids between fibroblasts from XP and a hamster line, followed by analysis of these cells for their capacity to survive and repair DNA after exposure to ultraviolet. Methods for initiation and propagation of skin fibroblasts from two subjects, male and female siblings with XP, have been described⁸. Details on the origin of the TG2 line of golden hamster fibroblasts, which has a non-reverting mutation in the gene for hypoxanthine-guanine phosphoribosyltransferase (HGPRT), the general hybridization procedure¹⁰ and methods for cell survival and DNA repair by unscheduled synthesis⁸ were also described previously. Hybrids were produced by fusion with Sendai virus and selected by ultraviolet irradiation followed by culture on HAT medium (Fig. 1).     

Amounts of Epstein-Barr Virus DNA in Somatic Cell Hybrids Between Burkitt Lymphoma-Derived Cell Lines

The amounts of Epstein-Barr virus DNA in somatic cell hybrids between human lymphoid cell lines were found to be higher than in the parental cell lines, although the hybrids were not induced to spontaneous virus production.     

Acceleration of Chloroplast Division in Somatic Cell Hybrids between Mesophyll and Cell Culture Protoplasts

Tobacco mesophyll protoplasts were fused with protoplasts fromcultured cells by electric fusion. When the fusion productswere cultured for 2 d, chloroplast division was observed inthe heterokaryocytes under fluorescence microscopy after stainingwith DAPI, while such chloroplast division was not observedwhen mesophyll protoplasts alone were cultured in the same condition. ³Permanent address: Research Institute of House Food IndustryCo. Ltd., Mikuriya Sakaemachi, Higashi-Osaka-shi, Osaka, 577Japan. (Received June 21, 1988; Accepted November 22, 1988)     

Human–Mosquito Somatic Cell Hybrids induced by Ultraviolet-inactivated Sendai Virus

WE have recently achieved interphylum heterokaryon and hybrid formation from human (HeLa, clone 5 of S3, courtesy Dr Grace Leidy)¹ and mosquito (Aedes aegyptae L., courtesy of the late Dr E. C. Suitor, Naval Medical Research Institute, Bethesda, Maryland)² cell lines, using ultraviolet-inactivated Sendai virus to induce fusion³.     

Rescue of Epstein-Barr Virus from Somatic Cell Hybrids of Burkitt Lymphoblastoid Cells

A Burkitt lymphoblastoid cell line, P3J-HR-1, was fused and hybridized to a human sternal marrow cell line. The somatic cell hybrids were negative when examined for Epstein-Barr virus (EBV) markers. When the hybrid cells were exposed to 5-iododeoxyuridine, both EBV-specific antigens and virus particles were induced as determined by the immunofluorescence test and by electron microscopy. The data presented suggest that the EBV genome can be transferred from a lymphoblastoid cell to another cell type during cell hybridization, that the EBV genome can persist in the hybrid cells for long periods of time, and that synthesis of the virus can be induced in the heterokaryons.     

Linkage Relationships of Seventeen Human Gene Loci as determined by Man-Mouse Somatic Cell Hybrids

Electrophoretic studies of the enzymes produced by sixty-four independently derived hybrid clones have made possible the detection of autosomal linkages on human chromosomes.     

Multiple Human <Emphasis Type="Italic">T K</Emphasis> Chromosomes in Human-Mouse Somatic Cell Hybrids

Selection treatment can enrich a population of human-mouse hybrid cells for those cells containing multiple copies of the thymidine kinase chromosome.     

Properties of Somatic Cell Hybrids Between Mouse Cells and Simian Virus 40-Transformed Rat Cells

Hybrids between mouse cells and simian virus 40 (SV40)-transformed rat cells were made, and their properties and chromosome constitution were investigated over many generations. Their hybrid nature was confirmed by enzyme studies. During a period of 1 year a loss of 10 to 20% of the total number of chromosomes was observed. The SV40 tumor antigen was present and remained present in the hybrids. The parental and hybrid cells were studied for agglutination with concanavalin A, for growth in soft agar, and for serum requirement. These growth and surface characteristics of the transformed cells appeared in the hybrids.     

Loss of Wild-Type ATRX Expression in Somatic Cell Hybrids Segregates with Activation of Alternative Lengthening of Telomeres

Alternative Lengthening of Telomeres (ALT) is a non-telomerase mechanism of telomere lengthening that occurs in about 10% of cancers overall and is particularly common in astrocytic brain tumors and specific types of sarcomas. Somatic cell hybridization analyses have previously shown that normal telomerase-negative fibroblasts and telomerase-positive immortalized cell lines contain repressors of ALT activity, indicating that activation of ALT results from loss of one or more unidentified repressors. More recently, ATRX or DAXX was shown to be mutated both in tumors with telomere lengths suggestive of ALT activity and in ALT cell lines. Here, an ALT cell line was separately fused to each of four telomerase-positive cell lines, and four or five independent hybrid lines from each fusion were examined for expression of ATRX and DAXX and for telomere lengthening mechanism. The hybrid lines expressed either telomerase or ALT, with the other mechanism being repressed. DAXX was expressed normally in all parental cell lines and in all of the hybrids. ATRX was expressed normally in each of the four telomerase-positive parental cell lines and in every telomerase-positive hybrid line, and was abnormal in the ALT parental cells and in all but one of the ALT hybrids. This correlation between ALT activity and loss of ATRX expression is consistent with ATRX being a repressor of ALT.     

Identification of Neurofibromatosis 1 (NF1) Homologous Loci by Direct Sequencing, Fluorescence in Situ Hybridization, and PCR Amplification of Somatic Cell Hybrids

Using fluorescence in situ hybridization (FISH), we have identified seven NF1-related loci, two separate loci on chromosome 2, at bands 2q21 and 2q33-q34, and one locus each on five other chromosomes at bands 14q11.2, 15q11.2, 18p11.2, 21q11.2-q21, and 22q11.2. Application of PCR using NF1 primer pairs and genomic DNA from somatic cell hybrids confirmed the above loci, identified additional loci on chromosomes 12 and 15, and showed that the various loci do not share homology beyond NF1 exon 27b. Sequenced PCR products representing segments corresponding to NF1 exons from these loci demonstrated greater than 95% sequence identity with the NF1 locus. We used sequence differences between bona fide NF1 and NF1 -homologous loci to strategically design primer sets to specifically amplify 30 of 36 exons within the 5′ end of the NF1 gene. These developments have facilitated mutation analysis at the NF1 locus using genomic DNA as template.     

哺乳动物体细胞核移植中供体细胞的研究进展

在哺乳动物体细胞核移植中,供体细胞是影响其效率的主要因素之一。供体细胞的类型、细胞周期、细胞的培养代数、冷藏与冷冻处理,以及供体动物的性别、年龄等都可能影响核移植胚胎的发育。根据现有资料,简要综述了在哺乳动物体细胞核移植中有关供体细胞的研究进展。