Construction and identification of recombinant lentivirus-mediated gene transfer system for rat transducer of regulated CREB activity 1

Objective： To construct a recombinant lentivirus vector which carries SD rat transducer of regulated CREB activity-1（TORC1） gene and examine its ability to express the TORC 1 gene in vitro. Methods： The coding sequence of SD rat TORC 1 gene was amplified using PCR and cloned into pGC-FU vector. 293T cells were transfected using Lipofectamine 2000 and packaged for the recombinant lentivirus particles. When the cloned sequence was identified to be right, the recombinant lentivirus particles were amplified in a large quantity. The titer of virus was determined by real-time PCR and the level of TORC1 expression was examined by Western blot. Results： The recombinant lentivirus vector carrying TORC1 was constructed successfully and could express TORC1 at a high level in 293T cells in vitro, and the titer determined by real-time PCR was 2 × 10^8 TU/ml. Conclusion： The recombinant lentivirus vector could express TORCl gene at a high level, and was very helpful in the study of exploring the effect of TORC1 on spinal cord injury.

An efficient strategy for generation of transgenic mice by lentiviral transduction of male germline stem cells in vivo

Background： Male germline stem cells（MGSCs） are a subpopulation of germ cells in the testis tissue. MGSCs are capable of differentiation into spermatozoa and thus are perfect targets for genomic manipulation to generate transgenic animals.Method： The present study was to optimize a protocol of production of transgenic mice through transduction of MGSCs in vivo using lentiviral-based vectors. The recombinant lentiviral vectors with either EF-1 or CMV promoter to drive the expression of enhanced green fluorescent protein（e GFP） transgene were injected into seminiferous tubules or inter-tubular space of 7-day-old and 28-day-old mouse testes. At 5 or 6 wk post-surgery, these pre-founders were mated with wild-type C57BL/6J female mice（1.5 to 2.0-month-old）.Results： Sixty-seven percent of F1 generation and 55.56 % of F2 offspring were positive for eG FP transgene under the control of EF-1 promoter via PCR analysis. The transgenic pups were generated in an injection site-and age-independent manner. The expression of transgene was displayed in the progeny derived from lentiviral vector containing CMV promoter to drive transgene, but it was silenced or undetectable in the offspring derived from lentiviral vector with transgene under EF-1 promoter. The methylation level of g DNA in the promoter region of transgene was much higher in the samples derived lentiviral vectors with EF-1 promoter than that with CMV promoter,suggesting e GFP transgene was suppressed by DNA methylation in vivo.Conclusion： This research reported here an effective strategy for generation of transgenic mice through transduction of MGSCs in vivo using lentivirus vectors with specific promoters, and the transgenic offspring were obtained in an injection site-and age-independent manner. This protocol could be applied to other animal species, leading to advancement of animal transgenesis in agricultural and biomedical fields.

Construction and identification of an RNA interference lentivirus vector targeting the Ras homology C gene of melanoma cells

Background Melanoma has the highest mortality among all superficial malignant tumors.The poor prognosis is due to its high metastasis rate and the lack of therapeutic targets.As a molecular switch that controls tumor metastasis,Ras homology C （RhoC） has been correlated with tumor progression,especially tumor invasion and metastasis.However,little research has been done about the effects of RNA interference （RNAi） targeting RhoC on the invasion and metastasis of melanoma.In this study,we constructed an RNAi lentivirus vector targeting the RhoC gene of melanoma cells and identified its silencing effects on the RhoC gene.Methods Based on the RhoC gene encoding information,three pGPU6/GFP/Neo-short hairpin （shRNA） plasmids were constructed.After detecting their silencing effects on the RhoC gene of A375 cells,the most effective pGPU6/GFP/ Neo-shRNA plasmid was packed with lentivirus to construct the recombinant pLenti6.3-EGFP-453 targeting RhoC.The lentivirus vector was used to infect A375 cells,and then the expression of RhoC mRNA and protein were determined with real-time PCR and Western blotting.Results The plasmids pGPU6/GFP/Neo-shRNA 336,pGPU6/GFP/Neo-shRNA 453,and pGPU6/GFP/Neo-shRNA 680 were constructed.After they were transfected into A375 cells,the expressions of RhoC mRNA and protein were 1.47±0.26,1.13±0.16,1.39±0.11 and 70.98±9.21,50.67±6.06,65.77±4.06,respectively.pGPU6/GFP/Neo-shRNA 453 was the most effective sequence,and was used to successfully construct the pLenti6.3-EGFP-453 lentiviral vector targeting RhoC.pLenti6.3-EGFP-453 was used to infect A375 cells.The expression of RhoC mRNA and protein were 1.05±0.05 and 62.04±15.86 in the lentivirus group,4.21±0.24 and 220.86±24.07 in the negative lentivirus control group,and 4.63±0.32 and 257.39±12.30 in the normal control group respectively with the difference between the lentivirus group and the control groups being statistically significant （P ＜0.05）.Conclusion The successfully constructed pLenti6.3-EGFP-453 vector targeting the RhoC can effectively infect human melanoma A375 cells in vitro,and significantly inhibit the RhoC mRNA and protein expression.

In vivo Inhibitory Effect of Lentivirus-mediated RNA Interference Targeting RhoC on Growth of SKOV3 Cells

To investigate the inhibitory effect of lentivirus-mendiated RNA interference targeting RhoC on the growth of SKOV3 cells（ovarian cancer SKOV3 cells） in vivo, the vector expressing RNA interference targeting RhoC gene（LV-shRhoC） was constructed and the virus particles were packaged. The infection effiency of SKOV3 cells by the virus was estimated by green fluorescent protein expression on a fluorescence microscope and the expression of RhoC gene in the SKOV3 cells was detected by reverse transcription real time polymerase chain reaction（PCR）. Furthermore, human ovarian cancer SKOV3 cells, empty vector infected SKOV3 cells and interfered-vector infected SKOV3 cells were respectively seeded into nude mice, and the shape, mass, volume and histophathological changes of the transplanted tumors were observed 20 d later the mice were sacrified. The results show that lentivirus packa-ging particles can effectively infect SKOV3 cells and the lentivirus-mediated RNA interference can significantly in- hibit the expression of RhoC gene in SKOV3 ceils, the mass and volume of the transplanted tumor in the mice of the specific-control group（Lv-shRhoC） are all lower than the corresponding ones in the mice of negative- and blank-control groups（Lv-NC and SKOV3）. Moreover, the histopathlosical secion investigation shows that the nuclear Karyotype and histopathologic mitotic figure of SKOV3 cells in mice of the specific-control group are clearly lower than those in the mice of the negative-control group.Thus it is concluded that silencing RhoC gene by means of lenti- virus-mediated RNA interference targeting RhoC can obviously inhibit the growth of ovarian cancer cells（SKOV3） in vivo, which is a new strategy for the gene therapy of ovarian cancers.

Construction and identification of lentiviral RNA interference vector of rat leptin receptor gene

Leptin resistance is a main mechanism of acquired childhood obesity,and the suppression of long form of leptin receptor(OBRb)gene expression in diet-induced obese rats indicates that the down-regulation of OBRb gene expression plays a pivotal role in the mechanism of leptin resistance.The aim of the present study was to construct the lentiviral RNA interference(RNAi)vector of rat OBRb gene and evaluate the effects of siRNA on silencing OBRb gene expression.The target sequence of siRNA-OBRb was designed,and the com-plementary DNA containing both sense and antisense oligonucleotides was synthesized.After phosphorylation and annealing,these double-stranded DNA was cloned to pRNA-lentivector-VGFP to construct pRNA-Lenti-OBRb-VGFP recombinants with U6-containing promoter,target sequence and Poly III terminator.Then,the products were confirmed by electrophoresis and sequencing analy-sis,and the effects of RNAi on reducing gene expression were further confirmed by real-time polymerase chain reaction in transfected rat glioma cells expressing OBRb.The target sequence of siRNA-OBRb was successfully cloned to pRNA-lentivector-VGFP,and the RNAi protocol specifically reduced the expression of OBRb mRNA by approximately 80%compared with controls in transfected rat glioma cells.The successful construction of rat lentivirus vectors expressing OBRb-specific shRNA may be useful for further investigation in vivo.

Roles of FOXP3 in experimental atherosclerosis of ApoE-knockout mice

Background Subtypes of T cells, called regulatory T cells （Treg cell）, play a critical role in limiting autoimmune processes and inflammatory responses, The aim of this study was to explore functional roles of FOXP3 in the manifestation of atherosclerosis in Apolipoprotein E deficient （ApoE）-/- mice. Methods Lentivirus-mediated （siRNA） was used to knock down FOXP3 and FOXP3high＋CD4＋ CD25＋ T cells adoptive transfer assays in high fat diet ApoE-/- mice were done. The resulting atherosclerotie lesions were assessed by determining FOXP3 transcript levels and investigating the expression of FOXP3 protein in different tissues. Results Animals treated with siRNA of FOXP3 showed a significant increase in atherosclerotic lesion formation and a reduction in the number of FOXP3＋CD4＋CD25＋ T cells compared with other groups. Transfer of FOXP3highCD4＋CD25＋ T cells significantly decreased atherosclerotic plaque formation and increased the number of FOXP3＋ CD4＋ CD25＋ T cells. FOXP3 protein levels and FOXP3 transcript levels were lowest in the siRNA group, and were highest in tissues from the Treg transfer group. Conclusion FOXP3 plays an important role in regulating the inflammatory response within the atherosclerotic lesion. It can inhibit significantly the progression of the atherosclerosis plaque in ApoE-/- mice.