Making BAC transgene constructs with lambda-red recombineering system for transgenic animals or cell lines.

The genomic DNA libraries based on Bacteria Artificial Chromosomes (BAC) are the foundation of whole genomic mapping, sequencing, and annotation for many species like mice and humans. With their large insert size, BACs harbor the gene-of-interest and nearby transcriptional regulatory elements necessary to direct the expression of the gene-of-interest in a temporal and cell-type specific manner. When replacing a gene-of-interest with a transgene in vivo, the transgene can be expressed with the same patterns and machinery as that of the endogenous gene. This chapter describes in detail a method of using lambda-red recombineering to make BAC transgene constructs with the integration of a transgene into a designated location within a BAC. As the final BAC construct will be used for transfection in cell lines or making transgenic animals, specific considerations with BAC transgenes such as genotyping, BAC coverage and integrity as well as quality of BAC DNA will be addressed. Not only does this approach provide a practical and effective way to modify large DNA constructs, the same recombineering principles can apply to smaller high copy plasmids as well as to chromosome engineering.

Recombineering-based procedure for creating BAC transgene constructs for animals and cell lines.

The use of BAC/P1 as a vector for the generation of a transgene has gained popularity after the genomic annotation of many organisms was completed (often based on the respective BAC library). Large-scale generation of BAC transgenic mice has proven that BAC transgene approaches have less integration position effects and dosage artifacts when compared with traditional transgenic approaches. Also, a BAC can achieve the same tissue-specific expression as a knock-in of the same gene with less effort and shorter time of establishment. The λ-RED recombinogenic system has been used to manipulate DNA constructs with site-directed mutagenesis, truncation, and tagging with an epitope tag or as a fusion protein by homologous recombination, as well as used here to modify many BACs with various transgenes. The recombineering plasmid, pKD46, is used to fabricate BAC transgenic constructs that can be used in generating transgenic organisms as well as used in mammalian cell culture.

Transforming growth factor beta mediates hepatocyte apoptosis through Smad3 generation of reactive oxygen species.

TGFbeta induces hepatocyte apoptosis via reactive oxygen species (ROS) generation, the mitochondrial permeability transition (MPT), and caspase activation. The role of the Smad pathway in these events is unknown. In this study primary hepatocytes were isolated from Smad3 wild-type (+/+) and knockout (-/-) mice, and were treated with TGFbeta (5ng/ml) and/or trolox (2mM). ROS generation, MPT, TGFbeta-dependent transcription, and apoptosis were assessed in the presence or absence of Smad3 wild-type (WT) and dominant-negative (DN) plasmids. With TGFbeta treatment, Smad3 (-/-) hepatocytes did not generate ROS activity, exhibit MPT, activate caspases, or undergo apoptosis when compared to Smad 3 (+/+) hepatocytes. Similarly, transfection of Smad3 (+/+) hepatocytes with DN-Smad3 inhibited TGFbeta-mediated transcription, ROS generation, MPT, and apoptosis. However, Smad3 (-/-) cells transfected with WT-Smad3 and treated with TGFbeta demonstrated increased transcriptional activity, the MPT, and TGFbeta-induced apoptosis. TGFbeta-mediated ROS generation occurred through an NADPH-like oxidase pathway since diphenyleneiodonium chloride inhibited ROS induction. In conclusion, TGFbeta-induced hepatocyte apoptosis occurs through Smad3 dependent activation of ROS with subsequent activation of the MPT and caspases.

Ski promotes tumor growth through abrogation of transforming growth factor-beta signaling in pancreatic cancer.

OBJECTIVE: We hypothesized that human pancreatic cancer resists TGF-beta signaling and cell death through increased Ski expression. SUMMARY BACKGROUND DATA: Ski is an oncogenic protein that acts as a TGF-beta repressor and prevents related gene transcription. Previous work suggests that Ski acts as an oncoprotein in melanoma and esophageal cancer. Ski expression and function have not been determined in human pancreatic cancer. METHODS: Immunohistochemistry and immunoblots assessed Ski expression in human pancreatic cancer. Panc-1 cells were treated with or without Ski siRNA, and Ski and Smad protein expression, transcriptional reporter activation, and growth assays were determined. Panc-1 cells were inoculated in the flank of nude mice and tumor volume and histology assessed after administration of Ski siRNA or control vector. RESULTS: Ski was abundantly expressed in human pancreatic cancer specimens assessed by immunohistochemistry (91%) and immunoblot analysis (67%). Panc-1 cells exhibited nascent Ski expression that was maximally inhibited 48 hours after transfection with Ski siRNA. TGF-beta transcriptional activity was increased 2.5-fold in Ski siRNA-treated cells compared with control (P < 0.05). Ski siRNA increased TGF-beta-induced Smad2 phosphorylation and p21 expression. Panc-1 growth in culture was decreased 2-fold at 72 hours. A Ski siRNA expression vector injected into nude mice resulted in a 5-fold decrease in growth. CONCLUSION: Inhibition of Ski through RNA interference restored TGF-beta signaling and growth inhibition in vitro, and decreased tumor growth in vivo.

Basal reactive oxygen species determine the susceptibility to apoptosis in cirrhotic hepatocytes.

Hepatocytes from cirrhotic murine livers exhibit increased basal ROS activity and resistance to TGFbeta-induced apoptosis, yet when ROS levels are decreased by antioxidant pretreatment, these cells recover susceptibility to apoptotic stimuli. To further study these redox events, hepatocytes from cirrhotic murine livers were pretreated with various antioxidants prior to TGFbeta treatment and the ROS activity, apoptotic response, and mitochondrial ROS generation were assessed. In addition, normal hepatocytes were treated with low-dose H(2)O(2) and ROS and apoptotic responses determined. Treatment of cirrhotic hepatocytes with various antioxidants decreased basal ROS and rendered them susceptible to apoptosis. Examination of normal hepatocytes by confocal microscopy demonstrated colocalization of ROS activity and respiring mitochondria. Basal assessment of cirrhotic hepatocytes showed nonfocal ROS activity that was abolished by antioxidants. After pretreatment with an adenovirus expressing MnSOD, basal cirrhotic hepatocyte ROS were decreased and TGFbeta-induced colocalization of ROS and mitochondrial respiration was present. Treatment of normal hepatocytes with H(2)O(2) resulted in a sustained increase in ROS and resistance to TGFbeta apoptosis that was reversed when these cells were pretreated with an antioxidant. In conclusion, cirrhotic hepatocytes have a nonfocal distribution of ROS. However, normal and cirrhotic hepatocytes exhibit mitochondrial localization of ROS that is necessary for apoptosis.

Primary cirrhotic hepatocytes resist TGFbeta-induced apoptosis through a ROS-dependent mechanism.

BACKGROUND/AIMS: The cirrhotic liver manifests dysregulated hepatocyte growth by poor regenerative capacity, formation of regenerative nodules, and malignant transformation to hepatocellular carcinoma. The purpose of this study was to determine if dysregulated hepatocyte growth occurs through deficient apoptosis. METHODS: Hepatocytes were isolated from normal and CCl(4)-treated mice and treated with TGFbeta, TNFalpha, and UV-C, known apoptotic agents. RESULTS: Cirrhotic hepatocytes were less sensitive to TGFbeta- (45+/-5 vs. 15+/-3%; P<0.003), TNFalpha- (59+/-21 vs. 21+/-8%; P=0.02), and UV-C-induced (31+/-4 vs. 17+/-4%; P<0.03) apoptosis compared to normal hepatocytes. In normal hepatocytes, TGFbeta-induced apoptosis occurred through a ROS-, MPT-, and caspase-dependent pathway. Cirrhotic hepatocytes lacked caspase activation, had decreased procaspase-8 expression, failed to undergo the MPT, and had increased basal ROS activity compared to normal hepatocytes. After treatment with trolox, an antioxidant that reduced basal ROS activity, cirrhotic hepatocytes underwent apoptosis in response to TGFbeta treatment. CONCLUSIONS: These findings suggest that increased ROS activity in cirrhotic hepatocytes plays a critical role in mediating cirrhotic hepatocyte resistance to apoptosis. Cirrhotic hepatocyte resistance to TGFbeta-induced apoptosis is ROS-dependent and is a mechanism of dysregulated growth in the chronically inflamed liver.