Biomedical subjects

T S Sidén

Publications and source records attributed to T S Sidén.

3 recordsLinked to original sources

PubMed1992

Radiation fusion hybrids for human chromosomes 3 and X generated at various irradiation doses.

We have used a gamma-irradiation (2.5-25 krads) cell fusion procedure to generate human-hamster somatic cell hybrids (IHB, irradiated human fragments in B14-150 cells), retaining small fragments derived from human chromosomes 3 and X. By using Alu-element mediated PCR amplification and dot-blot hybridization with human alphoid or total human DNA as probes, 86 positive hybrids were identified and selected for further analysis. Nonisotopic fluorescence in situ hybridization (FISH) with human DNA in a set of eight hybrids demonstrated the presence of from one to eight human fragments per cell independent of irradiation dose. In contrast, a significant dose-dependent variation of fragment sizes was shown in the analysis of the 86 hybrids with markers previously mapped to 3p (seven markers) and to Xq (21 markers). Using the Xq27-28 region as a model, 40% of the hybrids generated at 5 krads or less were found to have retained fragments in the range of 3-30 Mb, 10% retained the whole chromosome arm, and the remaining 50% retained fragments of less than 2-3 Mb. The proportion of fragments of 3 Mb or larger decreased rapidly at higher irradiation doses and was very low (less than 6%) in hybrids generated at 25 krads. Upon further characterization, the 86 hybrids analyzed here will provide a mapping panel for the entire chromosomes 3 and X with an estimated resolution in the range of 1-2 Mb on average, a size range amenable to PFGE and YAC contig mapping.

Animals↗

PubMed1990

Monochromosomal mouse microcell hybrids containing inserted selectable neo genes.

Normal mouse fibroblasts at early passage levels were used as a starting material to construct mouse-hamster microcell hybrids (MCH). The neor gene, carried on the pSV2neo and pZIP-NeoSV(X)1 plasmids, was introduced into the mouse fibroblasts by gene transfection and retroviral infection, respectively, prior to microcell hybridization into the E36 Chinese hamster cell line. In total about 180 MCH clones were isolated and their amount of mouse DNA was estimated by dot-blot analysis. About 50% of the transfection based hybrids (T-hybrids) showed signals indicating one mouse chromosome, less than 10% more than one mouse chromosome, and the remaining clones contained only subchromosomal amounts of mouse DNA. In the infection-based hybrid series (I-hybrids) more than 95% showed only subchromosomal mouse DNA content. Chromosomal integration analysis verified the presence of neor insertions in all 42 hybrid clones analyzed. C-banding analysis verified 14 of 15 hybrids scored as monochromosomals on dot blots. Chromosome fragmentation in T-type MCH was found to be (1) nonrandom, preferentially occurring in MCH derived from certain transfectants, (2) late in clonal establishment, and (3) essentially not related to prolonged cultivation in vitro. Once established, most T-type MCH clones including mono- and subchromosomal hybrids were essentially stable during prolonged cultivation. In contrast MCH initially containing several mouse chromosomes tend to lose the nonselectable ones during prolonged cultivation. In total we estimate the number of independent monochromosomal MCH derived in this study to more than 30.

Animals↗

PubMed1989

Construction of microcell hybrid panel containing different neo gene insertions in mouse chromosome 17 used for chromosome-mediated gene transfer.

A panel of four microcell hybrids representing different sites of insertion of the exogenous neo gene into mouse chromosome 17 has been constructed. These constructions were based on a cotransfer of mouse chromosome 17 and neomycin resistance generated in a stepwise procedure involving (1) random insertion of the neo gene into a primary cell hybrid containing mouse chromosome 17 in a hamster cell background, (2) microcell-mediated chromosome transfer (MMCT) to segregate mouse and hamster chromosomes, and (3) identification of the mouse chromosome containing cells using a novel cell dotting procedure for mass screening at the cell colony level by molecular hybridization. Using this panel of four microcell hybrids for chromosome mediated gene transfer (CMGT), we obtained one transformant containing a chromosome fragment derived from the t-complex region located on mouse chromosome 17. It is concluded that the specific chromosome based procedure used here to generate CMGT transfectants may provide a general means to produce large numbers of transfectants containing megabase fragments covering, in principle, all regions of a given chromosome.

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