Desert diversity: genome sequence of Gordonia rubripertincta cluster DJ phage Mossy and cluster DV phage Erutan.

Lytic bacteriophages Mossy and Erutan were directly isolated from desert soil on Gordonia rubripertincta and characterized by their morphologies and genomes. Mossy, part of the DJ cluster of Actinobacteriophage, has a genome of 61,183 bp. The genome of Erutan, part of the DV cluster, is 66,957 bp.

Genome Sequences of Gordonia rubripertincta Phages LilyPad and PokyPuppy.

Two lytic phages infecting Gordonia rubripertincta were isolated from irrigated desert soil. Phage LilyPad and PokyPuppy have 64,158-bp and 77,065-bp genomes, respectively. Based on gene content similarity to phages in the Actinobacteriophage database, LilyPad is assigned to phage subcluster DG1 and PokyPuppy to subcluster CS2.

Small fragment homologous replacement-mediated modification of genomic beta-globin sequences in human hematopoietic stem/progenitor cells.

An ultimate goal of gene therapy is the development of a means to correct mutant genomic sequences in the cells that give rise to pathology. A number of oligonucleotide-based gene-targeting strategies have been developed to achieve this goal. One approach, small fragment homologous replacement (SFHR), has previously demonstrated disease-specific genotypic and phenotypic modification after introduction of small DNA fragments (SDFs) into somatic cells. To validate whether the gene responsible for sickle cell anemia (beta-globin) can be modified by SFHR, a series of studies were undertaken to introduce sickle globin sequences at the appropriate locus of human hematopoietic stem/progenitor cells (HSPCs). The characteristic A two head right arrow T transversion in codon 6 of the beta-globin gene was indicated by restriction fragment length polymorphic (RFLP) analysis of polymerase chain reaction (PCR) products generated by amplification of DNA and RNA. At the time of harvest, it was determined that the cells generally contained

Limited restoration of cystic fibrosis lung epithelium in vivo with adult bone marrow-derived cells.

RATIONALE: Recent literature suggests that adult bone marrow-derived cells can localize to lung and acquire immunophenotypic characteristics of lung epithelial cells. We speculated this might be a potential therapeutic approach for correcting defective lung epithelium in cystic fibrosis. OBJECTIVE: To determine whether adult bone marrow-derived cells containing normal cystic fibrosis transmembrane conductance regulator protein (CFTR) could repopulate lung epithelium in transgenic mice deficient in that protein. METHODS: Stromal marrow cells or total marrow obtained from adult male wild-type mice were transplanted into adult female Cftr knockout mice. To increase marrow cell recruitment naphthalene was used to induce airway epithelial injury in recipient mice. MEASUREMENTS AND MAIN RESULTS: At 1 wk, 1 mo, and 3 mo after transplantation, Cftr mRNA was detected in lung homogenates of recipient mice by reverse transcription-polymerase chain reaction. Cftr mRNA was not found in either donor marrow cells or mature circulating leukocytes. In situ examination of recipient mouse lungs demonstrated rare (0.025%) chimeric airway epithelial cells, some of which (0.01%) expressed CFTR protein. Naphthalene-induced airway remodeling nonsignificantly increased the number of chimeric airway epithelial cells expressing Cftr. CONCLUSIONS: These results demonstrate that adult marrow cells can be recruited to airway epithelium and induced to express Cftr in mice otherwise lacking this protein. However, the number of observed chimeric epithelial cells is small and new strategies for enhancing airway epithelial remodeling by adult bone marrow-derived cells will be necessary for correction of defective CFTR-dependent chloride transport.

Acute lung injury with endotoxin or NO2 does not enhance development of airway epithelium from bone marrow.

Adult marrow-derived stem cells can localize to lung and acquire immunophenotypic characteristics of lung epithelial cells. Lung injury increases recruitment of the marrow-derived cells. We speculated that comparing patterns of lung engraftment following different lung injuries would provide insight into potential mechanisms by which marrow-derived cells were recruited to lung. To evaluate this, adult female C57Bl/6 mice irradiated and engrafted with marrow from adult male transgenic GFP mice were exposed to either intranasal inhalation of endotoxin (25 microg/mouse) or 3 days of 25 ppm NO(2) and then compared 1 or 3 months later to transplanted but otherwise uninjured mice. In all cases, the majority of marrow-derived cells recruited to lung were CD45(+) leukocytes. In lungs of transplanted but otherwise uninjured mice, small numbers of CD45(-) donor-derived cells in alveolar septae stained positively for pro-surfactant protein C. Rare donor-derived cells located in the airway epithelium stained positively with cytokeratin. Subsequent exposure of engrafted mice to NO(2) or endotoxin did not significantly increase the number or pattern of donor-derived CD45(-) cells found in recipient lungs. These results suggest that NO(2) or endotoxin lung injury does not result in significant engraftment of marrow-derived cells in lung.

Dual Y chromosome painting and in situ cell-specific immunofluorescence staining in lung tissue: an improved method of identifying donor marrow cells in lung following bone marrow transplantation.

Several recent studies have demonstrated localization of donor bone marrow-derived cells in recipient lungs following transplantation from male to female mice or patients. Donor cells are identified by detection of the Y chromosome by fluorescence in situ hybridization (FISH). However, protein digestion pretreatments usually required for tissue FISH significantly limit the ability to detect cell type-specific markers by immunohistochemistry. We have used an alternative protein digest approach that entails heating the slides in 10 mM sodium citrate rather than utilizing a protease digestion. This approach preserves cell proteins following FISH, and allows lung tissue to remain intact for subsequent detection of cell-specific markers by immunohistochemistry. We have examined this technique in mouse lungs using a Y chromosome paint and three cell-specific markers, a pan-cytokeratin for epithelial cells, PECAM-1 for endothelial cells, and CD45 for leukocytes. Excellent visualization of both the Y chromosome and cell-specific surface protein markers was obtained on a single slide. This approach will significantly enhance the ability to detect and identify donor bone marrow cells in recipient mouse lungs following male to female transplantation.

Construction and purification of pSABR 01, a pUC19-derived vector optimized for cloning full-length cDNA.

A novel pUC19-derived vector, pSABR 01, was constructed by sub-cloning a fragment of the pSPORT1 polylinker into PUC19. The insertion of the polylinker generated two inactivating mutations in the LacZ open reading frame. These were then repaired by a PCR-based Site Directed Mutagenesis strategy. The pSABR 01 plasmid has four sites that are recognized by 'rare-cutter' restriction endonucleases that will optimize the cloning of full-length cDNA and five dual restriction sites that increase the versatility of subcloning the inserted cDNA. Protocols were also defined for purification of pSABR 01 from residual pSPORT1, following pSABR 01 construction, and from another contaminating plasmid.

Sequence-specific modification of genomic DNA by small DNA fragments.

Small DNA fragments have been used to modify endogenous genomic DNA in both human and mouse cells. This strategy for sequence-specific modification or genomic editing, known as small-fragment homologous replacement (SFHR), has yet to be characterized in terms of its underlying mechanisms. Genotypic and phenotypic analyses following SFHR have shown specific modification of disease-causing genetic loci associated with cystic fibrosis, beta-thalassemia, and Duchenne muscular dystrophy, suggesting that SFHR has potential as a therapeutic modality for the treatment of monogenic inherited disease.