Construction of recombinant adenoviral vector carrying the LEDGFp52 gene by homologous recombination in bacteria and its expression in vitro / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To construct recombinant adenoviral vector carrying the LEDGFp52 gene by homologous recombination in bacteria and to detect its expression in vitro.METHODS: The LEDGFp52 gene was cloned to adenoviral shuttle plasmid pAdTrack-CMV. Then, the resultant pAdTrack-CMV-LEDGFp52 was cotransfected into BJ5 183 bacteria with the adenoviral backbone plasmid pAdeasy-1. The adenoviral plasmid carrying LEDGFp52 was generated with homologous recombination in bacteria, and the adenoviruses were produced in 293 cells. These 293 cells were then infected with adenoviruses, and the expression of LEDGFp52 was detected by CPE (cytopathic effect) and western blot.RESULTS: The titer of Ad-LEDGFp52 adenoviruses was up to 5×1012 pfu/L after proliferation in 293 cells. LEDGFp52 was expressed efficiently in 293 cells after infection.CONCLUSION: The recombinant adenoviruses vector expressing LEDGFp52 was constructed successfully and can be used in further gene transfection experiments.

Subject The Effect of Apolipoprotein E Overexpression on Plasma Lipoprotein Profile in Mice Fed on Long-term High Cholesterol Diet

BACKGROUND AND OBJECTIVES: Apolipoprotein E (apoE), a 34-kD plasma glycoapolipoprotein, plays a key role in lipoprotein metabolism by facilitating cellular uptake of remnants of triglyceride-rich chylomicrons and VLDL and may have other important biological functions. Various studies using apoE-knockout mice have elucidated the role of apoE in lipolysis, remnant clearance, and atherogenesis. Despite the growing evidence of the protective role exerted by apoE against atherosclerosis, the direct in vivo effects of the apoE overexpression on lipoprotein metabolism in the presence of endogenous mouse apoE are not yet fully understood. In this study, the technique of adenovirus-mediated gene transfer was employed to investigate the in vivo effect of apoE overexpression on lipid level and lipoprotein profile in mice fed on normal chow or high cholesterol diet. MATERIALS AND METHODS: Recombinant adenovirus (rAd.mApoE) containing mouse apoE cDNA driven by a cytomegalovirus promoter was generated and infused via tail vein in mice fed on normal chow or high cholesterol diet. Recombinant adenoviruses have emerged as the most efficient vectors for transient delivery of functional genes to the mammalian liver. RESULTS: rAd.mApoE in the various mouse tissues one week after injection was expressed mainly in the liver. ApoE overexpression decreased the cholesterol and triglyceride concentration in mice fed on normal chow. In mice fed on high cholesterol diet, apoE overexpression resulted in decrease in triglyceride concentration and increase in cholesterol. VLDL and LDL fraction were decreased, HDL was increased by apoE overexpression in both mice fed on normal chow and high cholesterol diet. CONCLUSION: These data suggest that overexpression of mouse apoE in mice with endogenous apoE may exert antiatherogenic effect by inducing favorable change in the lipoprotein profile, regardless of diet and consequent plasma lipid level. In the future, the studies regarding the effect of human apoE overexpression on the lipid and lipoprotein profile in mice fed on normal chow and high cholesterol diet will be helpful to understand the species differences or similarities in apoE activity.

RESEARCH ON BONE MARROW MESENCHYMAL STEM CELLS MODIFIED BY NT-3 GENE AND PRETREATED WITH RETINOIC ACID TO DIFFERENTIATE INTO NEURON-LIKE CELLS IN THE INJURED SITE OF SPINAL CORD / 解剖学报

Objective To explore the potential of bone marrow mesenchymal stem cells(MSCs) to be modified by neurotrophin-3(NT-3) gene and pretreated with retinoic acid(RA) to differentiate into neuron-like cells in the transplanted site of the spinal cord injury. Methods MSCs,RA-induced MSCs,LacZ gene modified MSCs,NT-3 gene modified MSCs,and MSCs both modified by NT-3 gene and pretreated with RA were immediately transplanted respectively into the completely transected site(T_(10) spinal segment) of spinal cord.On the 67th day after the operation,the spinal cord segment was removed and frozenly sectioned.The differentiation potential of MSCs was examined by immunofluorescence histochemistry and the percentage was calculated of neuron-like cells that were differentiated from MSCs among all the transplanted cells groups. Results Transplanted MSCs could differentiate into neural stem cells(nestin-positive), neuroglial cells(GFAP-positive) and neuron-like cells(NF and MAP2-positive) in the injured spinal cord.Some of them also differentiated into the neuronlike cells which contained some neurotransmitters(ChAT and 5-HT positive) or had the potential to form a synapse(PSD95-positive).The percentage of neuron-like cells differentiated from MSCs modified by NT-3 gene and pretreated with RA was the highest among all the transplanted cell groups.Conclusion MSCs modified by NT-3 gene and pretreated with RA could better to differentiate into neuron-like cells in the injured spinal cord.