A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference.

RNA interference (RNAi) has recently emerged as a specific and efficient method to silence gene expression in mammalian cells either by transfection of short interfering RNAs (siRNAs; ref. 1) or, more recently, by transcription of short hairpin RNAs (shRNAs) from expression vectors and retroviruses. But the resistance of important cell types to transduction by these approaches, both in vitro and in vivo, has limited the use of RNAi. Here we describe a lentiviral system for delivery of shRNAs into cycling and non-cycling mammalian cells, stem cells, zygotes and their differentiated progeny. We show that lentivirus-delivered shRNAs are capable of specific, highly stable and functional silencing of gene expression in a variety of cell types and also in transgenic mice. Our lentiviral vectors should permit rapid and efficient analysis of gene function in primary human and animal cells and tissues and generation of animals that show reduced expression of specific genes. They may also provide new approaches for gene therapy.

Genetic susceptibility to chronic hepatitis is inherited codominantly in Helicobacter hepaticus-infected AB6F1 and B6AF1 hybrid male mice, and progression to hepatocellular carcinoma is linked to hepatic expression of lipogenic genes and immune function-associated networks.

Helicobacter hepaticus causes hepatitis in susceptible strains of mice. Previous studies indicated that A/JCr mice are susceptible and C57BL/6NCr mice are resistant to H. hepaticus-induced hepatitis. We used F1 hybrid mice derived from A/J and C57BL/6 matings to investigate their phenotype and determine their hepatic gene expression profile in response to H. hepaticus infection. F1 hybrid mice, as well as parental A/J and C57BL/6 mice, were divided equally into control and H. hepaticus-infected groups and euthanized at 18 months postinoculation. Hepatic lesions were evaluated histologically and the differential hepatic gene expression in F1 mice was determined by microarray-based global gene expression profiling analysis. H. hepaticus-infected parental strains including A/J and C57BL/6 mice, as well as F1 mice, developed significant hepatitis. Overall, hepatocellular carcinomas or dysplastic liver lesions were observed in 69% of H. hepaticus-infected F1 male mice and H. hepaticus was isolated from hepatic tissues of all F1 mice with liver tumors. Liver tumors, characterized by hepatic steatosis, developed in livers with high hepatitis scores. To identify gene expression specific to H. hepaticus-induced hepatitis and progression to hepatocellular carcinoma in F1 mice, a method using comparative group transcriptome analysis was utilized. The canonical pathway most significantly enriched was immunological disease. Fatty acid synthase and steaoryl-coenzyme A desaturase, the two rate-limiting enzymes in lipogenesis, were upregulated in neoplastic relative to dysplastic livers. This study suggests a synergistic interaction between hepatic steatosis and infectious hepatitis leading to hepatocellular carcinoma. The use of AB6F1 and B6AF1 mice, as well as genetically engineered mice, on a C57BL/6 background will allow studies investigating the role of chronic microbial hepatitis and steatohepatitis in the pathogenesis of liver cancer.

CD4(+)CD25(+) regulatory lymphocytes require interleukin 10 to interrupt colon carcinogenesis in mice.

Roles for host immune response in carcinogenesis are not well defined. Recent studies have shown that microbially driven inflammation can lead to colon cancer and that prior transfer of regulatory lymphocytes expressing CD4 and CD25 prevents the innate inflammatory events that lead to colon cancer in mice. To further examine the ability of regulatory lymphocytes to inhibit carcinogenesis, 129/SvEv Rag-2-deficient mice were inoculated by gastric gavage with Helicobacter hepaticus, an enteric bacterial pathogen of mice. Mice were then treated at 1, 3, or 12 months after infection with adoptive transfer of CD4(+)CD45RB(lo)CD25(+)-regulatory cells. Mice dosed with regulatory cells at 4 or 12 weeks after H. hepaticus infection had reduced severity of inflammatory bowel disease and significantly lower risk of colon cancer during the 8 month observation period, compared with infected mice that had not received cells. This suggested that regulatory cells were able to interrupt the ongoing innate immune events in the stepwise progression to cancer. Transfer of regulatory cells into chronically infected mice with established cancer reduced severity of colitis, epithelial dysplasia, and cancer, but did not eliminate all tumors. Regulatory cells lacking anti-inflammatory cytokine interleukin (IL)-10 were unable to inhibit inflammatory bowel disease, dysplasia, or cancer, showing that IL-10 was required for the protective effects of lymphocytes in this setting. Taken together, the data suggest that IL-10-mediated suppression of host innate inflammatory response was pivotal in interrupting carcinogenesis. Regulatory lymphocytes and cytokines may have implications for novel therapies for colon cancer in humans.

Identification of cholelithogenic enterohepatic helicobacter species and their role in murine cholesterol gallstone formation.

BACKGROUND & AIMS: Helicobacter spp are common inhabitants of the hepatobiliary and gastrointestinal tracts of humans and animals and cause a variety of well-described diseases. Recent epidemiologic results suggest a possible association between enterohepatic Helicobacter spp and cholesterol cholelithiasis, chronic cholecystitis, and gallbladder cancer. To test this, we prospectively investigated the effects of Helicobacter spp infection in cholesterol gallstone pathogenesis in the highly susceptible C57L/J mouse model. METHODS: Helicobacter spp-free adult male C57L mice were infected with several different enterohepatic Helicobacter spp or left uninfected and fed either a lithogenic diet or standard mouse chow for 8 and 18 weeks. At the conclusion of the study, bile was examined microscopically and diagnostic culture and polymerase chain reaction were performed. RESULTS: Mice infected with Helicobacter bilis or coinfected with Helicobacter hepaticus and Helicobacter rodentium and fed a lithogenic diet developed cholesterol gallstones at 80% prevalence by 8 weeks compared with approximately 10% in uninfected controls. Monoinfections with H hepaticus , Helicobacter cinaedi , and H rodentium gave a cholesterol gallstone prevalence of 40%, 30%, and 20%, respectively; the latter 2 groups did not differ significantly from uninfected animals. Neither infected nor uninfected mice fed a chow diet developed cholesterol gallstones. CONCLUSIONS: These findings, along with prior epidemiologic studies, suggest that Helicobacter spp play a major role in the pathophysiology of cholesterol gallstone formation in mice and perhaps humans.