Keratin 7 is expressed in simple epithelia but is expressed at low or undetectable levels in gastrointestinal epithelial cells. In the pancreas, it is present in ductal but not in acinar cells. K7 mRNA is overexpressed in pancreatic cancers. Here we use luciferase reporter assays to analyze the tissue-specific regulatory elements of murine keratin 7 (Krt7) promoter in vitro and in vivo. All elements required for appropriate cell and tissue specificity in reporter assays are present within the Krt7 -234 bp sequence. This fragment appears more selective to pancreatic ductal cells than the Krt19 promoter. GC-rich sequences corresponding to putative Sp1, AP-2 binding sites are essential for in vitro activity. Krt7-LacZ transgenic mice were generated to analyze in vivo activity. Sequences located 1.5 or 0.25 kb upstream of the transcription initiation site drive reporter expression to ductal, but not acinar, cells in transgenic mice. LacZ mRNA was detected in the pancreas as well as in additional epithelial tissues--such as the intestine and the lung--using both promoter constructs. An AdK7Luc adenovirus was generated to assess targeting selectivity in vivo by intravenous injection to immunocompetent mice and in a xenograft model of pancreatic cancer. The -0.25 kb region showed pancreatic selectivity, high activity in pancreatic cancers, and sustained transgene expression in xenografts. In conclusion, the krt7 promoter is useful to target pancreatic ductal adenocarcinoma cells in vitro and in vivo.
A Rhizobium sp. (Hedysarum coronarium) calcof luor dark (Cal-) mutant, named CallO, was obtained following TnSmob-insertion mutagenesis. It is affected in the synthesis of exopolysaccharide and presents an altered lipopolysaccharide that is not recognized by a polyclonal antibody against the lipopolysaccharide of the parental strain. The residual exopolysaccharide obtained from the mutant lacks galactose and the high-molecular-mass acidic fraction. This mutant was complemented by plasmid pD56 that restores the production of exopolysaccharide, the alteration of lipopolysaccharide and the Cal+ phenotype. The data presented indicate that the gene in which the mutant is defective is homologous to the exoB gene of Rhizobium meliloti and fails to synthesize UDP-glucose 4-epimerase. The CallO mutant was Fix+ on H. coronarium (sulla) although it develops an indeterminate type of nodule, indicating that exopolysaccharide is not essential for a successful nodulation in this symbiotic association.
From 106 human blastocyts donate for research after in vitro fertilization (IVF) and preimplantation genetic diagnosis (PGD) for monogenetic disorder, 3 human embryonic stem cells (hESCs) HVR1, HVR2 and HVR3 were successfully derived. HVR1 was assumed to be genetically normal, HVR2 carrying Becker muscular dystrophy and HVR3 Hemophilia B. Despite the translocation t(9;15)(q34.3;q14) detected in HVR2, all the 3 cell lines were characterised in vitro and in vivo as normal hESCs lines and were registered in the Spanish Stem Cell Bank.
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