Mice lacking Ren1c were generated using C57BL/6-derived embryonic stem cells. Mice homozygous for Ren1c disruption (Ren1c؊/؊) are born at the expected ratio, but approximately 80% die of dehydration within a few days. The surviving

Duffy antigen/receptor for chemokines (DARC) is a promiscuous receptor for chemokines that is required for Plasmodium vivax infection of erythroid cells. This receptor is expressed by subsets of endothelial, as well as erythroid cells. Selection for protection from malaria infection resulted in an erythroid-specific defect, suggesting that DARC may play a critical role in endothelial biology. Mice with targeted disruption of this gene were generated, and the function of DARC in inflammation was explored. RNA from spleens of homozygous mutant mice lacked DARC transcripts, which were abundant in wild-type (+/+) and heterozygote (+/−) mice. DARC−/− mice lacked developmental abnormalities and were healthy at 1 year. Whereas hematologic parameters were within normal ranges, erythrocytes from nullizygous mice lacked CXC and CC chemokine-binding activity. Challenge with lipopolysaccharide resulted in significantly increased inflammatory infiltrates in lung and liver of nullizygous mice. These results suggest that DARC modulates the intensity of inflammatory reactions as a sink for chemokines.

To determine whether the expression of cardiac genes changes in a graded manner or by on/off switching when cardiac myocytes change genetic programs in living animals, we have studied two indicator genes that change their expression oppositely in mouse binucleate ventricular cardiomyocytes during development and in response to cardiac hypertrophy. One is a single-copy transgene controlled by an ␣-myosin heavy chain (aMHC) promoter and coding for CFP. The other is the endogenous ␤-myosin heavy chain (bMHC) gene modified to code for a YFP-bMHC fusion protein. Using high-resolution confocal microscopy, we determined the expression of the two indicator genes in individual cardiomyocytes perinatally and after inducing cardiac hypertrophy by transverse aortic constriction. Our results provide strong evidence that the cardiac genes respond by switching their expression in an on/off rather than graded manner, and that responding genes within a single cell and within the two nuclei of cardiomyocytes do not necessarily switch concordantly.gene switching ͉ myosin heavy chain

No abstract

Duffy antigen/receptor for chemokines (DARC) is a promiscuous receptor for chemokines that is required for Plasmodium vivax infection of erythroid cells. This receptor is expressed by subsets of endothelial, as well as erythroid cells. Selection for protection from malaria infection resulted in an erythroid-specific defect, suggesting that DARC may play a critical role in endothelial biology. Mice with targeted disruption of this gene were generated, and the function of DARC in inflammation was explored. RNA from spleens of homozygous mutant mice lacked DARC transcripts, which were abundant in wild-type (+/+) and heterozygote (+/−) mice. DARC−/− mice lacked developmental abnormalities and were healthy at 1 year. Whereas hematologic parameters were within normal ranges, erythrocytes from nullizygous mice lacked CXC and CC chemokine-binding activity. Challenge with lipopolysaccharide resulted in significantly increased inflammatory infiltrates in lung and liver of nullizygous mice. These results suggest that DARC modulates the intensity of inflammatory reactions as a sink for chemokines.

Progress in isolating stem cells from tissues, or generating them from adult cells by nuclear transfer, encourages attempts to use stem cells from affected individuals for gene correction and autologous therapy. Current viral vectors are efficient at introducing transgenic sequences but result in random integrations. Gene targeting, in contrast, can directly correct an affected gene, or incorporate corrective sequences into a site free from undesirable side effects, but efficiency is low. Most current targeting procedures, consequently, use positive-negative selection with drugs, often requiring >10 days. This drug selection causes problems with stem cells that differentiate in this time or require feeder cells, because the feeders must be drug resistant and so are not eliminated by the selection. To overcome these problems, we have developed a procedure for isolating gene-corrected stem cells free from feeder cells after 3-5 days culture without drugs. The method is still positive-negative, but the positive and negative drugresistance genes are replaced with differently colored fluorescence genes. Gene-corrected cells are isolated by FACS. We tested the method with mouse ES cells having a mutant hypoxanthine phosphoribosyltransferase (Hprt) gene and grown on feeder cells. After 5 days in culture, gene-corrected cells were obtained free from feeder cells at a ''purity'' of >30%, enriched >2,000-fold and with a recovery of Ϸ20%. Corrected cells were also isolated singly for clonal expansion. Our FACS-based procedure should be applicable at small or large scale to stem cells that can be cultured (with feeder cells, if necessary) for >3 days.electroporation ͉ flow cytometry ͉ fluorescent protein ͉ gene targeting ͉ gene therapy H ematopoietic stem cells (HSC) are among the longest known and best studied stem cells (1). They are characterized by their ability to fully repopulate the bone marrow of suitably prepared recipients and have been used therapeutically in the form of bone marrow transplants for several decades. HSC are, however, difficult to culture ex vivo without differentiating, and normal HSC have been induced to increase to only about six times the input number of HSC in vitro (2). The in vitro expansion of HSC from experimental animals has been enhanced (up to approximately forty times input) by the introduction of transgenes coding for MDR1 or HOXB4 (3, 4), but HSC cannot currently be clonally expanded in vitro. Their ability to selfreplicate extensively in vivo is, however, undoubted because a single HSC is sufficient to repopulate the bone marrow of a recipient, which can then be used to repopulate the bone marrows of secondary recipients (5, 6).The history of bone marrow transplantations clearly emphasizes that problems are likely to arise when there are histocompatibility differences between donors and recipients. Thus, the first successful bone marrow transplant was with an identical twin (genetically an autologous transplant) (7). Only the subsequent elucidation of histocompatibility antigens all...