Lentiviral Vector For RNA Interference Research Articles

Construction of lentiviral RNAi vector of PPARγ gene in cashmere goats and comparison with the transcriptome analysis of adipose cells treatments

As an important trait influencing meat quality, the deposition of Intramuscular fat (IMF) is affected by preadipocyte differentiation. Nuclear receptor peroxisome proliferator-activated receptorγ(PPARγ) plays an important role in the regulation of glucose homeostasis and lipid metabolism. In this study, lentivirus-mediated RNA interference (RNAi) was applied to inhibit PPARγ gene expression, which can explore the effects of PPARγ silencing on the preadipose of cashmere goats in vitro. The roles of PPARγ gene in proliferation and differentiation of adipocytes were investigated by MTT assay and Oil Red O staining extraction assay. Using Real-time PCR and Western blotting analyses, the gene expression levels in goat adipocytes were detected at 24, 36, 48 and 72 h after transfection. By the high-throughput sequencing, the comparative transcriptome analysis of four different samples including preadipocyte, adipocyte, shRNA- PPARγ intramuscular fat cell and control group in Cashmere goats was performed. The results showed that PPARγ gene could increase proliferation of goat adipocyte, but decrease their differentiation. The novel genes screened with 152, 450, 456, 412, 39 and 445 different expressions by high-throughput sequencing. The genes, such as PREF-1, PREF-1, FGF, TGF, TNF, RA, IL-21 and EGF had higher expression in shRNA-PPARγ intramuscular fat cells. The 10 pathways that related to adipocyte differentiation were discovered by GO and KEGG analysis. The most significant pathways involved in PPARγ, WNT, mTOR, and MAPK signaling. Some key gens including LPL, FABP, FATP, AP2, RXRG and CD36 were enriched in PPARγ signal pathway. All of these findings will provide a new reference for understanding adipose transcriptome and identifying novel pathways regulating adipose physiology.

Inhibition of X-linked inhibitor of apoptosis protein enhances anti-tumor potency of pure total flavonoids on the growth of leukemic cells.

Flavonoids, a vast group of polyphenols widely distributed in plants, are known to possess a range of biological activities and potential anti-tumor effects. X-linked inhibitor of apoptosis protein (XIAP) promotes the progression of leukemia by preventing tumor cells undergoing apoptosis. The present study investigated the potential effects and underlying mechanisms of pure total flavonoids from Citrus paradisi Macfad (PTFC) on human U937 cells, and explored the effects of short hairpin (sh)RNA-mediated XIAP knockdown on the anti-cancer effects of PTFC. Western blotting was used to determine level of apoptosis-associated effectors following PTFC treatment. A lentiviral vector of RNA interference of XIAP gene was constructed to downregulate XIAP expression. MTT assay and flow cytometry were used to determine the effects of PTFC separately or combined with XIAP-shRNA on inhibition and apoptosis of U937 cells, respectively. Treatment with PTFC effectively inhibited leukemic cell proliferation in a dose- and time-dependent manner. PTFC induced apoptosis of U937 cells in a dose-dependent manner, at a particular concentration range, by decreasing XIAP expression levels and activating caspases-3, −7 and −9. PTFC treatment combined with XIAP-shRNA additionally demonstrated a marked increase in cell apoptosis, compared with PTFC or XIAP-shRNA alone (P<0.05). Therefore, these findings suggest that PTFC inhibits growth and induces apoptosis in U937 cells in vitro. Furthermore, suppression of XIAP expression enhances these effects.

Construction and identification of a model for HJURP gene defect expression in human embryo villus cells

To construct a lentiviral vector for RNA interference (RNAi) of the HJURP gene and to identify the silencing efficiency in the human embryo villus cells and to provide a human embryo villus cells multiplication and chromosome segregation. In accordance with the study, three specific sequences of siRNA targeting HJURP gene were designed, synthesized, then the complementary DNA containing both sense and antisense oligonucleotides of the targeting sequences were annealed and inserted into the lentiviral vector.The correct clonings were confirmed by PCR and sequencing. The most effective recombinant lentivirus vector was screened, and the recombinant plasmids with the lentivirus packaging mixes were co-transfected into 293T cells to obtain packaged lentivirus particles. Then viral titer was determined. The silencing efficiency of target gene in human embryo villus cells was detected by Real-Time PCR. DNA sequencing showed that the shRNA sequence was successfully inserted into the lentivirus vector. The recombinant lentiviral vector was successfully transfected into 293T cells. The recombinant lentivirus had a titer of 108 PFU/ml. After silencing HJURP gene in human embryo villus cells, the expression level of HJURP mRNA decreased significantly and the RNAi efficiency was greater than 70%. A lentiviral shRNA expression vector targeting the HJURP gene was successfully constructed and may effectively silence the target gene at a cellular level, which provides a experimental model for the influence of HJURP gene expressing inhibition on human embryo villus cells multiplication and chromosome segregation.

Construction and identification of lentiviral vector mediated GATA-3 gene RNA interference in mouse

Objective:The aim of this study was to construct and identify a lentiviral vector for RNA interference of mouse GATA-3.Method:The coding region of the GATA-3 gene sequences were analyzed for a single-stranded oligo design and synthesis.PCR was performed for splicing reaction of synthetic oligo,which was synthesized into the lentiviral vector and transformed into the competent cell DH5α.An expression vector shRNA-GATA-3 was constructed after sequencing and identifying the gene sequence of the recombinant clones.Accoding to the target gene sequence ,four pairs are as following: siRNA-685 group(LV3-GATA-3-Mus-685),siRNA-1152 group (LV3-GATA-3-Mus-1152),siRNA-1615 group(LV3-GATA-3-Mus-1615) and control group ,which were designed and syntheized and builded into the lentiviral carrier.The shRNA vectors were cotransfected with expression vectors into the 293T cell line,and the optimal interference sequence was screened by QPCR. The 293T cells were cotranfected with the constructed lentiviral interference vectors and packing plasmids,the viruses were packed,the virus stock was collected and concentrated by ultracentrifugation,and the titer of the viruses was determined. Result:Gene sequence proved that the recombinant clone gene sequence was correct,and the lentiviral vector was successfully constructed. The relative expression of GATA-3 mRNA in siRNA-1615 group(0.004) was significantly reduced after RNA interference comparing with that in the control group(0.022),siRNA-1152 group(0.009) and siRNA-685 group(0.009). The screened LV3-GATA-3-Mus-1615 ,as the most effective interference sequence,was constructed into the lentiviral vector.The titer of the concentrated virus of LV3-GATA-3-Mus-1615 was 5×108 TU/ml in the viral supernate of 293T cells collected at 48 hours after co-transfection. Conclusion:Alentiviral RNAi expression vector targeting the GATA-3 gene (LV3-GATA-3-Mus-1615) was successfully constrcted and identified.

Effects of lentiviral vector‑mediated shRNA silencing of TGFβ1 on the expression of Col1a1 in rat hepatic stellate cells.

The present study aimed to construct a lentiviral RNA interference (RNAi) vector targeting the transforming growth factor β1 (TGFβ1) gene of rats, in order to examine its effect on silencing of the TGFβ1 gene and on the expression of collagen type 1 α1 (Col1a1) in HSC‑T6 rat hepatic stellate cells. Three RNAi sites of the TGFβ1 gene were selected according to its CDs sequence. Three pairs of small interfering RNA (siRNA) of these RNAi sites were synthesized and then transfected into HSC‑T6 cells, respectively, to confirm the optimal siRNA sequence via reverse transcription‑polymerase chain reaction analysis. Subsequently, shRNA targeting the sequence of the optimal siRNA was designed, synthesized and annealed to form a double‑stranded structure. The annealed oligonucleotide fragment was cloned into pGreenPuro plasmids to establish the pGreenPuro/TGFβ1 shRNA lentiviral vector, which was then transfected into 293T cells, following identification by restriction enzyme digestion and sequencing for the production of lentiviral particles exhibiting high reactivity. These particles were used to infect HSC‑T6 cells, following which the expression of GFP in the transfected cells was observed under an inverted microscope. The effects on TGFβ1 gene silencing and the expression levels of Colla1 were detected at the mRNA and protein levels. The results provided confirmation of the optimal siRNA sequence. Enzyme digestion and sequencing verified successful construction of the pGreenPuro/TGFβ1 shRNA lentiviral vector. This lentiviral vector effectively silenced the TGFβ1 gene in the HSC‑T6 cells, and inhibited the expression of Col1a1 at the mRNA and protein levels. Taken together, the lentiviral RNAi vector targeting the TGFβ1 gene of rats was successfully constructed, which effectively silenced the TGFβ1 gene of the HSC‑T6 cells and inhibited the expression of Col1a1.

Construction and identification of a lentiviral vector for RNA interference of human GLUT3 gene

To construct an effective lentiviral vector for RNA interference (RNAi) with human glucose transporter 3 (GLUT3)gene. Four pairs of shRNA sequences against different parts of GLUT3-mRNA were separately cloned into the RNAi plasmid vector pLV-shRNA by recombinant DNA technology to construct shRNA expression vectors pLV-shRNA-GLUT3-1, pLV-shRNA-GLUT3-2, pLV-shRNA-GLUT3-3, and pLV-shRNA-GLUT3-4. The vectors were transfected into HeLa cells to detect the effectiveness of GLUT3 gene silencing. One of effective vectors was selected and co-transfected into 293T cells with lentivirus packaging plasmids to obtain packaged lentivirus particles LV-GLUT3. After viral titer determination, U251 glioblastoma cells were infected with LV-GLUT3 at a multiplicity of infection (MOI) of 10. Finally, the expression of GLUT3 protein was detected by Western blot. DNA sequencing demonstrated that the shRNA sequences were successfully inserted into the pLV-shRNA vectors. In HeLa cells, the expression of GLUT3-mRNA was significantly down-regulated by the recombinant vectors compared with negative control. The recombinant lentivirus LV-GLUT3 harvested from 293T cells had a titer of 1.5×10(9) TU/mL. After infection with LV-GLUT3, the expression of GLUT3 protein in U251 glioblastoma cells was down-regulated. An effective lentiviral shRNA expression vector targeting the GLUT3 gene is successfully constructed and can be used for further study on the functions of GLUT3 gene.

The effect of lentiviral vector-mediated RNA interference targeting hypoxia-inducible factor 1α on the uptake of fluorodeoxyglucose ((18)f) in the human pancreatic cancer cell line, patu8988.

Hypoxia can stimulate (18)F-fluorodeoxyglucose ((18)F-FDG) uptake in cultured tumor cells. This study has investigated the effect of lentiviral vector-mediated RNA interference (RNAi) targeting hypoxia-inducible factor 1α (HIF-1α) on the changes in HIF-1 and glucose transporter 1 (Glut-1) expression, the cell growth, and the uptake of (18)F-FDG in the human pancreatic cancer cell line, Patu8988. Lentiviral RNAi vector targeting the HIF-1α gene (LV-HIF-1αRNAi) was constructed and used to treat cells at various concentrations (25-200 nM). The expression changes of HIF-1α and Glut-1 in hypoxic Patu8988 cells after RNAi treatment were determined using real time reverse transcription-polymerase chain reaction (real-time PCR). The inhibition rate of cell proliferation 48 hours after the addition of 10 μL of different concentrations of LV-HIF-1αRNAi (25-200 nM) was assayed using the MTT method. Meanwhile, the cell uptake of (18)F-FDG was also assessed. After RNAi transfection, the relative expression levels of HIF-1α mRNA and Glut-1 under hypoxia were reduced and the relative expression levels of HIF-1α protein also decreased. Compared with the control group, the inhibition rates of cell proliferation under different viral dosages were 5.98%, 15.65%, 26.42%, and 40.81%, respectively, positively correlated with the viral doses (r=0.558, p<0.05). Under hypoxia, Glut-1 mRNA expression in Patu8988 cells treated with 200 nM of LV-HIF-1αRNAi for 24, 48, and 72 hours, respectively, was positively correlated with the inhibition rate of cell proliferation (r=0.618, p<0.05) as well as the inhibition rate of (18)F-FDG uptake (r=0.664, p<0.05), while the latter two displayed a positive correlation with each other too (r=0.582, p<0.05). Under hypoxia, RNAi targeting HIF-1α significantly inhibited the expression of Glut-1 mRNA in Patu8988 pancreatic cancer cells and their uptake of (18)F-FDG. These results suggest that LV-HIF-1αRNAi may form a new treatment for pancreatic cancer, and the effectiveness of the treatment can be readily assessed with (18)F-FDG imaging.

RNA interference-mediated silencing of SOCS-1 via lentiviral vector promotes apoptosis of alveolar epithelial cells in vitro

Suppressor of cytokine signaling-1 (SOCS1) is a protein that negatively regulates cytokine and growth factor signaling. However, little is known regarding the precise role it plays in idiopathic pulmonary fibrosis. The aim of the present study was to construct a recombinant lentiviral vector for RNA interference targeting the SOCS1 gene and to detect the expression in human alveolar epithelial cells. A lentiviral vector-mediated RNA interference method was used to establish a SOCS1-negative cell line of alveolar origin (A549). Three pairs of complementary small hairpin RNA (shRNA) oligonucleotides targeting the SOCS1 gene were designed, synthesized and inserted into the pPll3.7 vector. Packaged lentivirus particles were obtained after 48 h, and the supernatant was used to transfect the human alveolar epithelial cell line A549. The expression of the SOCS1 protein was detected by Western blotting. MTT assay was used to detect the cell proliferation of alveolar epithelial cells with SOCS1 knockdown. The recombinant plasmids were confirmed by sequencing. The lentivirus-containing supernatant effectively infected the A549 cell line, and the expression of SOCS1 protein was inhibited, which was confirmed by Western blotting in the target cells. MTT assay indicated the inhibition effect for cell proliferation of A549 cells in the SOCS1-RNA interference group, compared to the control group with no interference-mediated silencing of the SOCS1 gene. A lentiviral vector for RNA interference targeting the SOCS1 gene was successfully constructed, and cell survival tests showed that knockdown of the SOCS1 gene promotes the apoptosis of alveolar cells.

Construction of lentiviral vector for RNA interference of Id2 gene and its effect on proliferation and apoptosis of gliomas cell line &lt;I&gt;in vitro&lt;/I&gt;

Construction of lentiviral vector for RNA interference of Id2 gene and its effect on proliferation and apoptosis of gliomas cell line &lt;I&gt;in vitro&lt;/I&gt;

Construction and identification of a recombinant lentiviral vector of RNA interference of lung cancer related gene SLC35F2

Objective To investigate the possibility of SLC35F2 inhibition by lentiviral vector-mediated RNA interference (RNAi) and the influence on cell proliferation and apoptosis in lung cancer cell line,and to set up a lung cancer cell line in which SLC35F2 is stably suppressed.Methods The lentiviral vector of siRNA targeted against SLC35F2 (SLC-siRNA) was constructed and transfected into the packaging cells 293T,and the viral supernatant was collected to transfect H1299 cells.After selection by puromycin and culture expansion,the stable cell clones were attained.Quantitative real-time fluorescent polymerase chain reaction (PCR) and Western blotting were used to detect the expression of SLC35F2.The effect of SLC35F2 silencing by RNAi on cell proliferation was quantified by CCK-8 assay.Annexin V-FITC/PI staining was employed to examine the apoptosis.Results Lentiviral vector SLC35F2-shRNA was constructed successfully.As compared with control group,the SLC35F2 expression was decreased to 81.8% in RNA and protein levels.CCK-8 revealed that the inhibition rate of H1299 cells transfected with SLC35F2 was 16.3%,and the apoptosis rate was significantly increased as compared with negative control group ( 14.88% vs 3.16% ,P ＜0.05 ).Conclusion Lentiviral vector-mediated RNA interference targeting against SLC35F2 can effectively inhibit SLC35F2 expression and cell proliferation.The lung cell line in which SLC35F2 gene was stably suppressed was successfully established. Key words: Long carcinoma; RNA interference; Lentiviral vector; Cell proliferation

Construction and identification of lentiviral vector for RNA interference targeting STUB1 gene

To construct and identification of a lentiviral vector for RNA interference (RNAi) targeting STUB1 gene. A pair of complementary small hairpin RNA (shRNA) oligonucleotides targeting STUB1 gene was designed, synthesized and inserted into linearized pMagic 4.0 vector. The recombinant plasmid was identified by double restriction digestion with Age I/EcoR I and DNA sequencing. PCR and DNA sequencing showed that the shRNA sequence was successfully inserted into pMagic 4.0 vector. The pMagic 4.0 vector was successfully packaged into lentivirus particles. A lentiviral shRNA expression vector and particles targeting STUB1 gene has been successfully constructed for the further study of the STUB1 gene.

Construction and identification of a lentiviral vector for RNA interference of the Cdx2 gene

Construction and identification of a lentiviral vector for RNA interference of the Cdx2 gene

Lentivirus-mediated SMO RNA interference inhibits SMO expression and cell proliferation, and affects the cell cycle in LNCaP and PC3 cancer cell lines

Smoothened (SMO) is an important member of the Hedgehog signaling pathway. We constructed a specific recombinant lentiviral vector for RNA interference, targeting the SMO gene (NM_005631) to observe its effect on SMO expression, cell proliferation and the cell cycle in the human androgen-sensitive prostate cancer cell line, LNCaP, and in the androgen-independent prostate cancer cell line, PC3. Four siRNA sequences were designed and inserted into a lentiviral vector pGCSIL-GFP to construct four recombinant vectors. The vector with the highest interfering efficiency was co-transfected with packaging vectors (pHelper1.0 and pHelper2.0) in 293T cells to assemble lentivirus particles by liposome for infecting LNCaP and PC3 cell lines, respectively. The expression level of SMO mRNA, tumor cell proliferation and cell cycle were measured by quantitative realtime polymerase chain reaction (qRT-PCR), 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay and flow cytometry, respectively. Sequence results showed that recombinant lentiviral vectors were constructed successfully. pGCSIL-GFP-723 had the highest interfering efficiency, named Lv-SIL-SMO723 after co-transfection, with which LNCaP and PC3 cell lines were infected. Compared with the control groups, results showed significantly decreased (P < 0.05) SMO mRNA expressions of LNCaP and PC3, lower mean percentage of S-phase cells and higher mean percentage of G(2)/M phase cells, as well as obviously slow proliferation (P < 0.01) of LNCaP in the infected group. Yet, the proliferation of PC3 was not altered (P > 0.05). In conclusion, the recombinant lentivirus particles were able to suppress SMO expression, regulate the cell cycle in the LNCaP and PC3 cell lines and markedly inhibit proliferation of LNCaP cells but not PC3 cells.

Construction and identification of lentiviral vector of RNA interference of RelB gene

Objective To construct a recombinant lentivirus plasmid of RNA interference (RNAi)of RelB gene.Methods According to the GenBank information of RelB,5 complementary DNA containing both sense and antisense Oligo DNA of the targeting sequences were designed,synthesized,and cloned into PlentiLox 3.7 which contained U6 promoter and green fluorescent protein(GFP).The positive clones were confirmed by PCR,sequencing,and authentication with KspA Ⅰand Hind Ⅲ.RM-1 cells were transfected and detected by Western-blot.293T cells were cotransfected with lentiviral vector and virus packaging plasmids.All virus stocks were produced by calcium phosphate mediated transfection.Results PCR and DNA sequencing demonstrated that the lentivirus RNAi vector of RelB was constructed successfully.The most effective siRNA sequence was 5'-TTGGAAATCATCGACGAAT-3'.The titer of concentrat ed virus was 8×1010 pfu/L.Conclusion The lentivirus RNAi vector of RelB was constructed successfully.The lentivirus RNAi vector of RelB significantly inhibited the RelB expression. Key words: siRNA; Lentiviral vector; Construction; Identification

Construction of a lentiviral vector for RNA interference of human VIM gene and its silencing effect in pancreatic cancer cells

Objective To construct a lentiviral expression vector for RNA interference (RNAi) of human VIM gene; and assess its gene silencing effect in pancreatic cancer cell line Panc-1.

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 dietinduced 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 complementary 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 con.rmed by electrophoresis and sequencing analysis, and the effects of RNAi on reducing gene expression were further con.rmed 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 speci.cally 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.

Construction and identification of lentiviral vector of the RNA interference of human CCL5 gene

Objective To construct a lentiviral vector of RNA interference of chemokine CC motif ligand-5 (CCLS) gene and identify its knockout effectiveness in Hela cells. Methods The pGCSIL-GFP plasmids which expressed four RNAi sequences ( a1, a2, a3, a4) respectively for human CCL5 gene togeth-er with pHelper 1.0 and Helper 2.0 plasmids were transfected into 293T cells in order to construct tentivi-ral vectors of RNA interference of CCL5 gene. Then the lentiviral vectors were transfected into Hela cells, and RT-PCR was used to evaluate its knockout effectiveness for CCL5. Results The knockout effective-ness of a1 ,a2 or a3 was beyond 95% excepting a4 ,whose knockout effectiveness was beyond 90% in Hela cells. Conclusion The knockout effectiveness of the constructed lentviral vectors for human CCL5 was satisfactory. Our results demonstrated that RNAi is able to silence the potential key gene CCL5 in human cancer cells. Key words: CCL5 gene ; RNA interference; Lentiviral vector; Construction; Knockout effectiveness

Fatty acid metabolism in adipocytes: functional analysis of fatty acid transport proteins 1 and 4

The role of fatty acid transport protein 1 (FATP1) and FATP4 in facilitating adipocyte fatty acid metabolism was investigated using stable FATP1 or FATP4 knockdown (kd) 3T3-L1 cell lines derived from retrovirus-delivered short hairpin RNA (shRNA). Decreased expression of FATP1 or FATP4 did not affect preadipocyte differentiation or the expression of FATP1 (in FATP4 kd), FATP4 (in FATP1 kd), fatty acid translocase, acyl-coenzyme A synthetase 1, and adipocyte fatty acid binding protein but did lead to increased levels of peroxisome proliferator-activated receptor gamma and CCAAT/enhancer binding protein alpha. Both FATP1 and FATP4 kd adipocytes exhibited reduced triacylglycerol deposition and corresponding reductions in diacylglycerol and monoacylglycerol levels compared with control cells. FATP1 kd adipocytes displayed an approximately 25% reduction in basal (3)H-labeled fatty acid uptake and a complete loss of insulin-stimulated (3)H-labeled fatty acid uptake compared with control adipocytes. In contrast, FATP4 kd adipocytes as well as HEK-293 cells overexpressing FATP4 did not display any changes in fatty acid influx. FATP4 kd cells exhibited increased basal lipolysis, whereas FATP1 kd cells exhibited no change in lipolytic capacity. Consistent with reduced triacylglycerol accumulation, FATP1 and FATP4 kd adipocytes exhibited enhanced 2-deoxyglucose uptake compared with control adipocytes. These findings define unique and distinct roles for FATP1 and FATP4 in adipose fatty acid metabolism.