Markers linked to the bc-3 gene conditioning resistance to bean common mosaic potyviruses in common bean

Necrotic strains of bean common mosaic potyviruses are becoming increasingly problematic in bean growing areas of Africa and Europe. Pyramiding epistatic resistance genes provides the most effective long-term strategy for disease control against all known strains of the virus. Indirect selection using tightly linked markers should facilitate the breeding of desired epistatic resistance gene combinations. In common bean, the most effective strategy for broad spectrum control of the bean common mosaic potyviruses is to combine I and bc-3 genes. We describe the use of near-isogenic lines and segregating populations from different gene pools combined with bulked segregant analysis to identify markers tightly linked with the recessive bc-3 gene that conditions resistance to all strains of bean common mosaic necrosis virus. We identified a RAPD marker, OG6595, linked at 3.7 cM from the bc-3, and the marker was used to confirm the location of bc-3 gene on bean linkage group B6. A codominant AFLP marker, EACAMCGG-169/172was identified and linked at 3.5 cM from the bc-3 and the AFLP and OG6595 markers flanked the bc-3 gene. The AFLP marker was converted to the STS marker SEACAMCGG-134/137 which showed co-segregation with the original AFLP marker. The 134 bp fragment associated with resistance was linked with the bc-3 gene present in a diverse group of bean genotypes except in two kidney bean lines. The OG6595 marker mapped on B6 supported independence of bc-3 from the I gene located on B2, which provides the opportunity to readily combine both genes in a single bean cultivar for broad spectrum resistance to bean common mosaic potyviruses

Intrahepatic injection of recombinant adeno-associated virus serotype 2 overcomes gender-related differences in liver transduction

The liver is an attractive organ for gene therapy because of its important role in many inherited and acquired diseases. Recombinant adeno-associated viruses (rAAVs) have been shown to be good candidates for liver gene delivery, leading to long-term gene expression. We evaluated the influence of the route of administration on rAAV-mediated liver transduction by comparing levels of luciferase expression in the livers of male and female mice after injection of rAAV serotype 2, using three different routes of administration: intravenous (IV), intraportal (IP), or direct intrahepatic (IH) injection. To determine transgene expression we used a noninvasive optical bioluminescence imaging system that allowed long-term in vivo analysis. After IV injection dramatic differences in liver transgene expression were observed, depending on gender. When IP injection was used the differences were reduced although they were still significant. Interestingly, direct intrahepatic injection of rAAV vectors was associated with the fastest and strongest onset of luciferase expression. Moreover, no gender differences in liver transduction were observed and luciferase expression was confined to the site of injection. Thus, direct intrahepatic injection of rAAV offers specific advantages, which support the potential of this route of administration for future clinical applications

Molecular therapy for obesity and diabetes based on a long-term increase in hepatic fatty-acid oxidation

Obesity-induced insulin resistance is associated with both ectopic lipid deposition and chronic, low-grade adipose tissue inflammation. Despite their excess fat, obese individuals show lower fatty-acid oxidation (FAO) rates. This has raised the question of whether burning off the excess fat could improve the obese metabolic phenotype. Here we used human-safe nonimmunoreactive adeno-associated viruses (AAV) to mediate long-term hepatic gene transfer of carnitine palmitoyltransferase 1A (CPT1A), the key enzyme in fatty-acid β-oxidation, or its permanently active mutant form CPT1AM, to high-fat diet-treated and genetically obese mice. High-fat diet CPT1A- and, to a greater extent, CPT1AM-expressing mice showed an enhanced hepatic FAO which resulted in increased production of CO(2) , adenosine triphosphate, and ketone bodies. Notably, the increase in hepatic FAO not only reduced liver triacylglyceride content, inflammation, and reactive oxygen species levels but also systemically affected a decrease in epididymal adipose tissue weight and inflammation and improved insulin signaling in liver, adipose tissue, and muscle. Obesity-induced weight gain, increase in fasting blood glucose and insulin levels, and augmented expression of gluconeogenic genes were restored to normal only 3 months after AAV treatment. Thus, CPT1A- and, to a greater extent, CPT1AM-expressing mice were protected against obesity-induced weight gain, hepatic steatosis, diabetes, and obesity-induced insulin resistance. In addition, genetically obese db/db mice that expressed CPT1AM showed reduced glucose and insulin levels and liver steatosis. Conclusion: A chronic increase in liver FAO improves the obese metabolic phenotype, which indicates that AAV-mediated CPT1A expression could be a potential molecular therapy for obesity and diabetes

Treatment of chronic viral hepatitis in woodchucks by prolonged intrahepatic expression of interleukin-12

Chronic hepatitis B is a major cause of liver-related death worldwide. Interleukin-12 (IL-12) induction accompanies viral clearance in chronic hepatitis B virus infection. Here, we tested the therapeutic potential of IL-12 gene therapy in woodchucks chronically infected with woodchuck hepatitis virus (WHV), an infection that closely resembles chronic hepatitis B. The woodchucks were treated by intrahepatic injection of a helper-dependent adenoviral vector encoding IL-12 under the control of a liver-specific RU486-responsive promoter. All woodchucks with viral loads below 10(10) viral genomes (vg)/ml showed a marked and sustained reduction of viremia that was accompanied by a reduction in hepatic WHV DNA, a loss of e antigen and surface antigen, and improved liver histology. In contrast, none of the woodchucks with higher viremia levels responded to therapy. The antiviral effect was associated with the induction of T-cell immunity against viral antigens and a reduction of hepatic expression of Foxp3 in the responsive animals. Studies were performed in vitro to elucidate the resistance to therapy in highly viremic woodchucks. These studies showed that lymphocytes from healthy woodchucks or from animals with low viremia levels produced gamma interferon (IFN-gamma) upon IL-12 stimulation, while lymphocytes from woodchucks with high viremia failed to upregulate IFN-gamma in response to IL-12. In conclusion, IL-12-based gene therapy is an efficient approach to treat chronic hepadnavirus infection in woodchucks with viral loads below 10(10) vg/ml. Interestingly, this therapy is able to break immunological tolerance to viral antigens in chronic WHV carriers

Molecular markers linked to the fin gene controlling determinate growth habit in common bean

Bulked segregant analysis (BSA) was used to identify molecular markers linked to determinate growth habit (fin gene) in a F2 population derived from the cross Andecha (FinFin) × BRB130 (finfin). Fourteen RAPD markers and one microsatellite (BMd45-AIA) were linked to the fin gene when tested on the entire population. The SCAR, SI19b, designed from the RAPD marker OI19385 (linked to the fin gene at a recombination fraction, RF = 0.14; LOD = 5.29), showed a codominant segregation in the F2 population Andecha × BRB130 consistent with that of the corresponding RAPD. The microsatellite BMd45-AIA showed a codominant segregation in this population and was also linked to the fin gene (RF = 0.11; LOD = 10.73). The DNA sequences of markers SI19b and BMd45-AIA present in Andecha and BRB130 were analyzed. The two alleles of marker SI19b differ in 4 single base pairs and in a deletion of 30 bp, whereas the two alleles of marker BMd45-AIA differ in a single base pair and in a deletion of 38 bp. In both cases, specific short sequences (CTGAAGA for SI19b and ACAGAGAGA for BMd45-AIA) were repeated at the beginning and the end of the deleted segments, suggesting that "looping" structures during replication could have caused these deletions. Linkage between the fin gene and the molecular markers SI19b and BMd45-AIA was investigated in two other segregating populations: a F2 population derived from the cross MDRK (finfin) × V225 (FinFin), and a RIL population derived from the cross Xana (finfin) × Cornell 49242 (FinFin). SI19b was polymorphic (codominant segregation) only in the RIL population Xana × Cornell 49242, and showed a weak linkage with the fin gene (26.9 cM; LOD = 2.34). BMd45-AIA showed codominant segregation in both populations, and was tightly linked to the fin gene, at 4.1 cM (LOD = 9.2) in the F2 population MDRK × V225, and at 3.3 cM (LOD = 16.2) in the RIL population Xana × Cornell 49242. © 2007 Springer Science+Business Media B.V.This work was supported by grants AGL2001-2676-CO2 and RTA02-052-C2 from the Ministerio de Ciencia y Tecnología, Spain. Astrid Pañeda was recipient of a salary fellowship from the Fundación para el Fomento de la Investigación Científica y Tecnológica (FICYT), Principado de Asturias, Spain. Cristina Rodríguez-Suárez was recipient of a salary fellowship from the Ministerio de Ciencia y Tecnología, Spain. Ana Campa was recipient of a salary fellowship from the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA, Spain).Peer reviewe