Michael R. James

Stable correction of a genetic deficiency in human cells by an episome carrying a 115 kb genomic transgene.

Persistent expression of a transgene at therapeutic levels is required for successful gene therapy, but many small vectors with heterologous promoters are prone to vector loss and transcriptional silencing. The delivery of genomic DNA would enable genes to be transferred as complete loci, including regulatory sequences, introns, and native promoter elements. These elements may be critical to ensure prolonged, regulated, and tissue-specific transgene expression. Many studies point to considerable advantages to be gained by using complete genomic loci in gene expression. Large-insert vectors incorporating elements of the bacterial artificial chromosome (BAC) cloning system, and the episomal maintenance mechanisms of Epstein–Barr virus (EBV), can shuttle between bacteria and mammalian cells, allowing large genomic loci to be manipulated conveniently. We now demonstrate the potential utility of such vectors by stably correcting a human genetic deficiency in vitro. When the complete hypoxanthine phosphoribosyltransferase (HPRT) locus of 115 kilobases (kb) was introduced into deficient human cells, the transgene was both maintained as an episome and expressed stably for six months in rapidly dividing cell cultures. The results demonstrate for the first time that gene expression from an episomal genomic transgene can correct a cell culture disease phenotype for a prolonged period.

Bipolar affective disorder partially cosegregates with a balanced t(9;11)(p24;q23.1) chromosomal translocation in a small pedigree

Analysis of an extended pedigree in which a balanced t(9;11)(p24;q23.1) translocation was found to cosegregate with bipolar affective disorder revealed that five of 11 translocation carriers had bipolar affective disorder and one carrier had unipolar depression. There were no affected individuals in the pedigree without the balanced translocation. We hypothesized that gene(s) or gene regulatory regions disrupted by the translocation might be contributing to the bipolar affective disorder in a dominant fashion. To test this hypothesis, we isolated the derivative chromosome 9 and derivative chromosome 11 in somatic cell hybrids and identified the nearest flanking markers on chromosome 9 (D9S230 and D9S2011E/HRFX3) and chromosome 11 (EST00652 and CRYA2). YAC contigs were constructed in the region of flanking markers for both chromosomes 9 and 11. Chromosome 11 breakpoint was localized within an 8-kb region in a small insert (100 kb) YAC. Chromosome 9 breakpoint was localized within approximately 2 Mb region. Several genes and ESTs including EST00652, CRYA2, DRD2, 5HTR3 on chromosome 11 and VLDLR and SLC1A1 on chromosome 9 were mapped within the vicinity of the breakpoint but were shown not to be disrupted by the translocation breakpoint. Although several possibilities exist regarding the role of the balanced translocation in developing bipolar affective disorder in this pedigree, including a chance cosegregation, identification of a disrupted gene or gene regulatory region with the help of physical mapping resources described in this study may help to identify the presence of a susceptibility gene for this disorder.

A whole-genome radiation hybrid panel and framework map of the rat genome

Linda C. McCarthy, * ** Marie-Therese Bihoreau,* Susanna L. Kiguwa,* Julie Browne, Takeshi K. Watanabe, Haretsugu Hishigaki, Atsushi Tsuji, Susanne Kiel, 2 Caleb Webber, Maria E. Davis, Catherine Knights, Angela Smith, Ricky Critcher, 1 Patrick Huxtall, 1 James R. Hudson, Jr., 4 Toshihide Ono, Hiroumi Hayashi, Toshihisa Takagi, Yusuke Nakamura, Akira Tanigami, 3 Peter N. Goodfellow, *** G. Mark Lathrop, 2 Michael R. James

Characterization of newly developed SSLP markers for the rat.

Abstract. We have isolated more than 12,000 clones containing microsatellite sequences, mainly consisting of (CA)n dinucleotide repeats, using genomic DNA from the BN strain of laboratory rat. Data trimming yielded 9636 non-redundant microsatellite sequences, and we designed oligonucleotide primer pairs to amplify 8189 of these. PCR amplification of genomic DNA from five different rat strains yielded clean amplification products for 7040 of these simple-sequence-length-polymorphism (SSLP) markers; 3019 markers had been mapped previously by radiation hybrid (RH) mapping methods (Nat Genet 22, 27–36, 1998). Here we report the characterization of these newly developed microsatellite markers as well as the release of previously unpublished microsatellite marker information. In addition, we have constructed a genome-wide linkage map of 515 markers, 204 of which are derived from our new collection, by genotyping 48 F2 progeny of (OLETFxBN)F2 crosses. This map spans 1830.9 cM, with an average spacing of 3.56 cM. Together with our ongoing project of preparing a whole-genome radiation hybrid map for the rat, this dense linkage map should provide a valuable resource for genetic studies in this model species.