Competitive priming PCR: a versatile method to generate cohesive terminus.

In the post-genomic era, providing a detailed description of protein functions poses a formidable challenge. To gain functional insights, we have to construct many kinds of expression vectors. DNA recombination based on polymerase chain reaction (PCR) and digestion followed by ligation is the preferred method for vector construction. However, this existing pattern is intrinsically limited by the selection of restriction endonuclease, prompting researchers to use commercial suppliers rather than amplifying genes themselves. Moreover, this method would introduce additional bases into the PCR products, which may be undesired for the construction of epitope expressing plasmid. A PCR-based gene synthesis method, referred to as competitive priming PCR (CP-PCR), is described here to efficiently assemble the plasmid expressing fusion protein of fibrinogen alpha chain and binding domain of galactose transcription factor. A pair of competitive sense primers were designed for the same target sequence. With the presence of antisense primer, PCR amplification of target sequence was performed in the same one system. The PCR product was underwent single digestion by using PstI, followed by ligation with the vector pCMV-BD linearized with EcoRIand PstI. The reconstructed plasmid was validated by sequencing and the fusion protein was affirmed by western blot. CP-PCR combines the superior convenience and precision of PCR. Moreover, it is perfectly capable of generating nearly all kinds of cohesive terminuses, which are ready to recombination in the presence of single digestion or even in the absence of digestion. We demonstrate, by using CP-PCR, the feasibility of directed cloning interested sequence only in the requirement of single digestion or even in the absence of digestion. Competitive priming PCR is demonstrated with convenience and precision equivalent to the traditional method. More than that, "seamless" DNA recombination may be achieved by this novel strategy.

Aldolase A-HBsAg interaction and its effect on ultraviolet radiation induced apoptosis in 293FT cells.

BACKGROUND AND AIM: Hepatitis B virus (HBV) infection poses great challenges to humans, claiming one million lives annually worldwide. Solid data have related HBV to hepatocellular carcinoma. METHODS: In the present research, we verified the interaction between surface protein (HBs) encoded by HBV and aldolase A (ALDA) using yeast two-hybrid, mammalian two-hybrid, co-immunoprecipitation, GST pull-down and laser scanning confocal. RESULTS: Anti-ALDA antibody precipitated Gal4-HBs fusion protein in the presence of HBs. Anti-HBs antibody precipitated p65ΔN-ALDA only in the presence of ALDA. Small HBs could be pulled down by GST-ALDA. Cells transfected with pCMV-AD-ALDA showed a protection from ultraviolet radiation-induced apoptosis (21.3% ± 1.3% for ALDA, 35.4% ± 2.1% for control, P < 0.05). CONCLUSIONS: An interaction does exist between ALDA and HBs. The S region within HBs is sufficient for binding ALDA. In addition, ALDA conferred protection to ultraviolet radiation-induced apoptosis, and this effect was enhanced by the interaction between HBs and ALDA.

Integrated application of uniform design and least-squares support vector machines to transfection optimization.

BACKGROUND: Transfection in mammalian cells based on liposome presents great challenge for biological professionals. To protect themselves from exogenous insults, mammalian cells tend to manifest poor transfection efficiency. In order to gain high efficiency, we have to optimize several conditions of transfection, such as amount of liposome, amount of plasmid, and cell density at transfection. However, this process may be time-consuming and energy-consuming. Fortunately, several mathematical methods, developed in the past decades, may facilitate the resolution of this issue. This study investigates the possibility of optimizing transfection efficiency by using a method referred to as least-squares support vector machine, which requires only a few experiments and maintains fairly high accuracy. RESULTS: A protocol consists of 15 experiments was performed according to the principle of uniform design. In this protocol, amount of liposome, amount of plasmid, and the number of seeded cells 24 h before transfection were set as independent variables and transfection efficiency was set as dependent variable. A model was deduced from independent variables and their respective dependent variable. Another protocol made up by 10 experiments was performed to test the accuracy of the model. The model manifested a high accuracy. Compared to traditional method, the integrated application of uniform design and least-squares support vector machine greatly reduced the number of required experiments. What's more, higher transfection efficiency was achieved. CONCLUSION: The integrated application of uniform design and least-squares support vector machine is a simple technique for obtaining high transfection efficiency. Using this novel method, the number of required experiments would be greatly cut down while higher efficiency would be gained. Least-squares support vector machine may be applicable to many other problems that need to be optimized.

Role of extracellular RNA and TLR3-Trif signaling in myocardial ischemia-reperfusion injury.

BACKGROUND: Toll-like receptor 3 (TLR3) was originally identified as the receptor for viral RNA and represents a major host antiviral defense mechanism. TLR3 may also recognize extracellular RNA (exRNA) released from injured tissues under certain stress conditions. However, a role for exRNA and TLR3 in the pathogenesis of myocardial ischemic injury has not been tested. This study examined the role of exRNA and TLR3 signaling in myocardial infarction (MI), apoptosis, inflammation, and cardiac dysfunction during ischemia-reperfusion (I/R) injury. METHODS AND RESULTS: Wild-type (WT), TLR3(-/-), Trif(-/-), and interferon (IFN) α/ß receptor-1 deficient (IFNAR1(-/-)) mice were subjected to 45 minutes of coronary artery occlusion and 24 hours of reperfusion. Compared with WT, TLR3(-/-) or Trif(-/-) mice had smaller MI and better preserved cardiac function. Surprisingly, unlike TLR(2/4)-MyD88 signaling, lack of TLR3-Trif signaling had no impact on myocardial cytokines or neutrophil recruitment after I/R, but myocardial apoptosis was significantly attenuated in Trif(-/-) mice. Deletion of the downstream IFNAR1 had no effect on infarct size. Importantly, hypoxia and I/R led to release of RNA including microRNA from injured cardiomyocytes and ischemic heart, respectively. Necrotic cardiomyocytes induced a robust and dose-dependent cytokine response in cultured cardiomyocytes, which was markedly reduced by RNase but not DNase, and partially blocked in TLR3-deficient cardiomyocytes. In vivo, RNase administration reduced serum RNA level, attenuated myocardial cytokine production, leukocytes infiltration and apoptosis, and conferred cardiac protection against I/R injury. CONCLUSION: TLR3-Trif signaling represents an injurious pathway during I/R. Extracellular RNA released during I/R may contribute to myocardial inflammation and infarction.

Interacting with HBsAg compromises resistance of jumping translocation breakpoint protein to ultraviolet radiation-induced apoptosis in 293FT cells.

Jumping translocation breakpoint protein (JTB) is suppressed in many cancers, implying it plays a role in the neoplastic transformation of cells. In order to explore the role of JTB in the carcinogenesis of liver, we used mammalian two-hybrid, co-immunoprecipitation, GST pull-down and laser scanning confocal to verify the interaction between HBs and JTB. According to the results, HBs interacts with JTB. In addition, we further determined that S region within HBs is sufficient for binding JTB. Overexpression of JTB conferred resistance to apoptosis induced by ultraviolet radiation, whereas this effect was compromised by the co-overexpression of HBs.