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Polymorphic differences altering expression of genes without changing their products probably underlie human quantitative traits affecting risks of serious diseases, but methods for investigating such quantitative differences in animals are limited. Accordingly, we have developed a procedure for changing the expression in mice of chosen genes over a 100-fold range while retaining their chromosomal location and transcriptional controls. To develop the procedure, we first dissected the effects in embryonic stem (ES) cells of elements within and downstream of the 3' untranslated region (UTR) of a single copy transgene at the Hprt locus. As expected, protein expression varied with the steady-state level and half-life of the mRNA. The rank order of expression with various tested 3' regions is the same in ES cells, and in cardiomyocytes and trophoblastocytes derived from them. In mice having two functionally different native genes with modified 3'UTRs, the desired expression was obtained.

No abstract

Summary The neuron cytoplasmic protein gene product 9·5 (PGP9·5)/ubiquitin‐C‐terminal hydrolase 1 (UCHL‐1) protein is a thiol protease that recognizes and hydrolyzes a peptide bond at the C‐terminal of ubiquitin, and is involved in the processing of ubiquitin precursors and ubiquinated proteins. Although this molecule is known as a specific tissue marker for the neuroendocrine system, many reports have indicated that PGP9·5 is a marker for certain tumour types, such as cancer of the lung, colon, and pancreas. The expression of PGP9·5 in myeloma cells was examined. PGP9·5 seemed to be expressed specifically in myeloma cells as compared with other haematological malignant cells. In addition, in myeloma cells subjected to growth‐factor starvation, the upregulation of PGP9·5 was observed in association with that of p27Kip1, a cyclin‐dependent‐kinase inhibitor, although the upregulation caused by irradiation was milder. In contrast, the hypoxic culture of myeloma cells induced down‐regulation of PGP9·5. These results suggested that PGP9·5 may be a good marker for myeloma among haematological malignancies. In addition, it may indicate certain cellular features of myeloma cells, such as sensitivity to proteasome inhibitors.

Homologous recombination (gene targeting) has many desirable features for gene therapy, because it can precisely correct mutant genes and restore their normal expression, and random nonhomologous integration of DNA is infrequent in cells in which homologous recombination has occurred. There are, however, no reports of attempts to use homologous recombination to correct mutant genes in normal hematopoietic stem cells (HSCs), which are prime cells for therapy of a variety of hematological and other conditions, presumably because of their low abundance and uncertainty that homologous recombination can occur at a usable frequency in these cells. The experiments reported here encourage optimism in this respect by demonstrating targeted correction of a defective hypoxanthine phosphoribosyltransferase gene in hematopoietic progenitor cells that can form colonies in methylcellulose culture. These clonogenic cells are in the same lineage as HSCs but are more abundant and more mature and so less pluripotent. T he ideal form of gene therapy would correct a mutant gene directly without causing changes elsewhere in the genome (1). Many of the problems associated with gene therapy would thereby be greatly reduced or eliminated, including lack of adequate expression, extinction of expression, and the mutagenesis associated with integrating the correcting sequences into random sites in the genome. Homologous recombination has the necessary prerequisites for use in this context, because it is capable of precisely correcting mutant genes (2), and random nonhomologous integration of targeting DNA into the genome is infrequent in cells in which homologous recombination has occurred (3). However, we find no reported attempts of using homologous recombination to correct mutant genes in normal hematopoietic stem cells (HSCs; ref. 4), which are prime cells for therapy of a variety of hematological and other conditions (5). The likely reasons appear to be the low abundance of these cells combined with uncertainty that homologous recombination can occur in them at a usable frequency. The experiments we report here were designed to test the possibility of using homologous recombination to correct a mutant gene in hematopoietic progenitor cells that can form colonies in tissue culture. These colony-forming cells (CFCs) are in the same lineage as HSCs but are more abundant and more mature, and therefore less pluripotent. We demonstrate that the hypoxanthine phosphoribosyltransferase (HPRT) gene, which is mutated in humans with Lesch-Nyhan disease (6, 7), can be corrected by homologous recombination in CFCs at frequencies equivalent to those seen in embryonic stem (ES) cells, encouraging optimism that homologous recombination to correct mutant genes in pluripotent stem cells capable of long-term hematopoietic repopulation eventually will prove feasible. . The mixtures were plated in standard methylcellulose medium (MethoCult GF M3434; StemCell Technologies, Vancouver) containing the following recombinant cytokines: mouse stem cell factor (50...