Interfering RNA Expression Vector Research Articles

Functional analysis of <italic>StMAPK4</italic> in response to low temperature stress in potato

<p id="C3">Potato is vulnerable to low temperatures resulting in reduced yield production. <italic>MAPK</italic> gene is widely involved in a variety of environmental stress, and it has been detected to be involved in low temperature regulation. In this present study, to explore <italic>StMAPK4</italic> function in response to low temperature stress, potato cultivar ‘Atlantic’ as the experimental material, the expression characteristics of <italic>StMAPK4</italic> gene were analyzed in potato root, stem, and leaf at the different time under low temperature (4℃) stress. <italic>StMAPK4</italic> gene was analyzed using bioinformatics and its encoded protein subcellular localization was assayed. <italic>StMAPK4</italic> overexpression and RNA interference expression vectors were constructed and obtained transgenic potato plants. The activities of superoxide dismutase (SOD) and peroxidase (POD), and the contents of proline (Pro) and malondialdehyde (MDA) in non-transgenic (NT), overexpressed and RNAi interfered transgenic plants were analyzed under 4℃. The results showed that the isoelectric point (pI) of StMAPK4 was 4.97 and it was acidic protein localized in the nucleus and cell membrane. The relative expression levels of <italic>StMAPK4</italic> in roots, stems, and leaves significantly increased under low temperature stress. Compared with non-transgenic plants, the activities of SOD and POD and the content of proline in <italic>StMAPK4</italic> overexpressed plants were significantly increased, while the content of MDA was significantly decreased. Compared with NT plants, the activities of SOD and POD, and the content of Pro in <italic>StMAPK4</italic> overexpression plants were significantly decreased, while the content of MDA was significantly increased. Phenotypic observation revealed that the leaves of non-transgenic and RNAi interfered expression plants wilted seriously, while the leaves of overexpressed plants were less affected. In summary, the overexpression of <italic>StMAPK4</italic> gene can enhance the tolerance of low temperature stress in potato plants.

The relationship between EuSRPP1 gene expression and rubber biosynthesis in Eucommia ulmoides Oliver (Du-zhong)

Because of the unique properties, trans-polyisoprene rubber (TPI) has gradually realized that it has great development potential. Eucommia ulmoides Oliver (E. ulmoides Oliver), also named Du-zhong, is one of the few woody plants producing TPI. The significant expansion of small rubber particle protein and rubber elongation factor (SRPP/REF) genes in E. ulmoides Oliver may be associated with the producing of TPI. To understand the relationship between EuSRPP1 gene expression and rubber biosynthesis in E. ulmoides Oliver, in this study, an overexpression transgenic (OT) vector of EuSRPP1 driven by the act promoter and an RNA interference (RNAi) expression vector of EuSRPP1 were constructed. Then, Agrobacterium-mediated genetic transformation of E. ulmoides Oliver was conducted to obtain the OT and RNAi seedlings. Quantitative Real-time Polymerase Chain Reaction (qRT-PCR) showed that the EuSRPP1 expression levels in OT seedlings significantly increased, and were 2.53 × and 3.68 × higher in OT-1 and OT-2 seedlings, respectively, than the wild-type (WT) seedlings, Meanwhile, the expression levels of E. ulmoides Isopentenyl Diphosphate Isomerase (EuIPPS) and E. ulmoides Farnesyl Diphosphate Synthase (EuFPS) genes in OT plants, which are related to rubber biosynthesis synthesis, were also up-regulated. To the contrary, EuSRPP1 gene expression levels in the RNAi-1 and RNAi-2 seedlings significantly decreased, which were 3.92% and 42.38% of the WT seedlings, respectively. EuIPPS and EuFPS genes expression levels in the RNAi seedlings were also down-regulated. The TPI content analysis revealed that the TPI content in the overexpression EuSRPP1 callus increased to 1.85 × that of the WT callus, whereas that in the RNAi callus decreased to only 52% of the WT callus. Molecular weights of rubber were significantly different in OT callus (2.31 × 106 Da) and RNAi callus (0.36 × 106 Da). Observation of the rubber particles revealed that the rubber particle electron density was higher in OT seedlings, and the immunogold localization results showed that EuSRPP1 was localized to rubber particles. These results indicate that the EuSRPP1 expression level influences the chain elongation, particle development, and content of rubber in E. ulmoides Oliver.

Construction of RNA interference (RNAi) lentiviral expression vector of DEK gene and its effect on the biological behavior of liver cancer cells

Objective: To construct RNA interference (RNAi) lentiviral expression vector of DEK gene, and to explore its effect on the biological behavior of liver cancer cells. Methods: Double-stranded oligo DNAs were annealed and synthesized according to the interference sequence of DEK gene by RNAi technology. Small interfering RNA expression vector pLKO.1 was cloned after enzymatic digestion. The recombinant lentiviral pLKO.1-sh hDEK was constructed, and then the virus supernatant was collected, packed and infected by 293T cells. Real-time reverse transcription PCR (RT-PCR) and Western blot were used to detect DEK expression in human liver cancer cells Bel-7402, Hu-7, SmMC-7721 and HepG2, and DEK knockdown efficiency in each group of lentivirus-infected cells. Cell proliferation ability, cloning ability, apoptosis and migration ability were detected by cell counting kit-8 (CCK8), flow cytometry and scratch test, respectively. The t-test was used to compare the mean between the two groups, and one-way ANOVA was used to compare the multiple groups. Results: Enzymatic digestion and DNA sequencing results confirmed that the recombinant lentiviral vectors pLKO.1-sh hDEK1 and pLKO.1-sh hDEK3 were successfully constructed. RT-PCR and Western blot results showed that the expression of DEK in human liver cancer cells BEL-7402 and Huh7 cells was higher, and pLKO.1-sh hDEK3 was more effective in inhibiting the DEK gene expression (P < 0.05). Therefore, pLKO.1-sh hDEK3 was selected to infect BEL-7402 and Huh7 cells for subsequent functional experiments. CCK8 cell proliferation test result showed that the cell proliferation ability of BEL-7402 and Huh7 cells infected with recombinant lentivirus was weakened when compared with blank control and negative control group (P < 0.05). Apoptosis results showed that the apoptosis rate of knockdown group was higher than that of blank and the negative control group (P < 0.05). Cell scratch test result showed that the wound healing rate of knockdown group was lower than that of blank control and negative control group (P < 0.05), and the difference was statistically significant; however, there was no statistically significant difference between blank control and negative control group. Conclusion: Targeting DEK expression in silent liver cancer cells can inhibit the cell proliferation, migration ability, and induce apoptosis, which lays the foundation for further study of the role of DEK gene in liver cancer.

Successful generation of anti-ToCV and TYLCV transgenic tomato plants by RNAi

Tomato is an economically important vegetable. Tomato chlorosis virus (ToCV) and Tomato yellow leaf curl virus (TYLCV) are two major viruses that cause serious losses to tomato production. The effective method to control these two viruses is to breed antiviral species by genetic engineering techniques. In order to obtain the RNA interference (RNAi) expression vector of tomato, the coat protein (CP) genes of ToCV and TYLCV were selected in this study. The tandem sequences of the two CP genes were obtained using the recombinant PCR technique. Using Gateway cloning technology, the RNAi expression vector pRNAi-ToCV-TY including the two CP genes was constructed by attB×attP and attL×attR recombination reactions. Polymerase chain reaction and sequencing analysis confirmed that the vector was obtained successfully and contained the ToCV and TYLCV CP genes. The RNAi expression vector pRNAi-ToCV-TY was transformed into Agrobacterium strain GV3101. The RNAi vector was then used to transform tomato. The objective fragments were successfully transformed into a tomato by PCR identification. At the fourth-leaf stage, the positive transgenic plants were challenged with ToCV and TYLCV. Out of 15 transgenic plants, 33 % showed early symptoms within 4 weeks post-infection (WPI); 20 % showed delayed symptoms (5 - 7 WPI); and the remaining 47 % were symptomless even after 9 WPI. The untransformed control plants (90 %) showed severe symptoms within 2 - 4 WPI, whereas 10 % delayed symptoms.

A novel plasmid-Escherichia coli system produces large batch dsRNAs for insect gene silencing.

RNA interference (RNAi)-based pest management requires efficient delivery and large-batch production of double-stranded (ds)RNA. We previously developed a nanocarrier-mediated dsRNA delivery system that could penetrate an insect's body and efficiently silence gene expression. However, there is a great need to improve the plasmid-Escherichia coli system for the mass production of dsRNA. Here, for efficient dsRNA production, we removed the rnc gene encoding endoribonuclease RNase III in E. coli BL21(DE3) and matched with the RNAi expression vector containing a single T7 promoter. The novel pET28-BL21(DE3) RNase III-system was successfully constructed to express vestigial (vg)-dsRNA against Harmonia axyridis. dsRNA was extracted and purified from cell cultures in four E. coil systems, and the yields of dsRNA in pET28-BL21(DE3) RNase III-, pET28-HT115(DE3), L4440-BL21(DE3) RNase III- and L4440-HT115(DE3) were 4.23, 2.75, 0.88 and 1.30 μg mL-1 respectively. The dsRNA expression efficiency of our novel E. coil system was three times that of L4440-HT115(DE3), a widely used dsRNA production system. The RNAi efficiency of dsRNA produced by our system and by biochemical synthesis was comparable when injected into Harmonia axyridis. Our system expressed dsRNA more efficiently than the widely used L4440-HT115(DE3) system, and the produced dsRNA showed a high gene-silencing effect. Notably, our pET28-BL21(DE3) RNase III-system provides a novel method for the mass production of dsRNA at low cost and high efficiency, which may promote gene function analysis and RNAi-based pest management. © 2020 Society of Chemical Industry.

Enhancement of grain number per spike by RNA interference of cytokinin oxidase 2 gene in bread wheat

BackgroundThis study aimed to validate the function of CKX gene on grain numbers in wheat.Methodswe constructed and transformed a RNA interference expression vector of TaCKX2.4 in bread wheat line NB1. Southern blotting analysis was used to select transgenic plants with single copy. The expression of TaCKX2.4 gene was estimated by Quantitative real-time PCR (qRT-PCR) analysis. Finally, the relation between expression of TaCKX2.4 gene and grain numbers was validated.ResultsTotally, 20 positive independent events were obtained. Homozygous lines from 5 events with a single copy of transformed gene each were selected to evaluate the expression of TaCKX2.4 and grain numbers per spike in T3 generation. Compared with the control NB1, the average grain numbers per spike significantly increased by 12.6%, 8.3%, 6.5% and 5.8% in the T3 lines JW39-3A, JW1-2B, JW1-1A and JW5-1A, respectively.ConclusionOur study indicated that the expression level of TaCKX2.4 was negatively correlated with the grain number per spike, indicating that the reduced expression of TaCKX2.4 increased grain numbers per spike in wheat.

Analysis of the peanut transgenic offspring with depressing AhFAD2 gene

The delta-12 fatty acid desaturase (Δ¹² FAD or FAD2) is a key enzyme that catalyzes oleic acid to linoleic acid by dehydrogenation at Δ¹² position of fatty acid carbon chain. In peanut, reduction or loss of FAD2 activity could enhance the relative content of oleic acid in kernels, and improve the quality and oxidation stability of peanut kernels and products. RNA interference (RNAi) technology could lead to non-expression or down-regulated expression of AhFAD2 gene. We constructed two RNA interference expression vectors with the inverted repeat sequence of partial AhFAD2 gene, which were driven separately by cauliflower mosaic virus (CaMV) 35S promoter or soybean agglutinin lectin seed-specific promoter. Homozygous transgenic lines carrying the two constructs stably in genetics were developed by peanut genetic transformation. There were no significant differences between the transgenic lines and the control through investigating the main agronomic traits. We analyzed the transcriptional level expression of AhFAD2 gene in transgenic lines and the control by real-time fluorescence quantitative PCR (qRT-PCR). The results suggested that the target genes of transgenic lines were likely suppressed by RNA interference, but showed different transcriptional levels in different peanut transgenic lines. Compared with untransformed lines, the resulting down-regulation of AhFAD2 gene resulted in a 15.09% or 36.40% increase in oleic acid content in the seeds of transformed HY23 and FH1 lines respectively, and the content of linoleic acid decreased by 16.19% or 29.81%, correspondingly, the ratio of oleic acid and linoleic acid (O/L) improved by 38.02%, 98.10%. The oleic acid content had significant differences between the two transformation constructs, and also among different transgenic lines. Moreover, the inhibition effect of RNAi was more obvious in the transgenic lines with FH1 as the receptor, and with transformation structure driven by seed specific promoter. The suppressed expression of AhFAD2 gene enabled the development of peanut fatty acid, which indicated that RNA interference would be a reliable technique for the genetic modification of peanut seed quality and the potential for improvement of other traits as well.

‘Psoriasis 1’ reduces psoriasis‑like skin inflammation by inhibiting the VDR‑mediated nuclear NF‑κB and STAT signaling pathways

‘Psoriasis 1’, a Chinese herbal medicine (CHM) formulation, is extensively used to treat psoriasis in China. Although this CHM formulation yields good therapeutic effect, the underlying mechanism of how this works remains unknown. The present study aimed to test the hypothesis that the CHM formulation ‘psoriasis 1’ inhibits vitamin D receptor (VDR)-mediated inflammation in psoriasis. To test this, a model of psoriasis was established by stimulating keratinocytes (HaCaT cells) with tumor necrosis factor (TNF)-α; these cells were subsequently transfected with a lentiviral VDR RNA interference expression vector. The expression levels of 25-hydroxyvitamin D3 (25HVD3), TNF-α, interleukin (IL)-4, IL-1, IL-17C, IL-23 and IL-6 were measured using ELISA, and the expression levels of VDR, inhibitor of nuclear factor (NF)-κB (IKK), NF-κB, signal transducer and activator of transcription (STAT) 3 and STAT4 were measured using reverse transcription-quantitative polymerase chain reaction analysis and western blotting. It was observed that ‘psoriasis 1’ downregulated the concentrations of TNF-α, IFN-γ, IL-22, IL-17C, IL-1β and IL-4, and upregulated the concentration of 25HVD3; furthermore, ‘psoriasis 1’ downregulated the expression levels of NF-κB, phosphorylated (p)-NF-κB, IKK, p-IKK, STAT3, p-STAT3, STAT4 and p-STAT4, and upregulated the expression level of VDR in TNF-α-induced HaCaT cells. These results suggested that ‘psoriasis 1’ suppressed the inflammatory response and the activation of the NF-κB and STAT signaling pathways. In addition, it was identified that silencing VDR expression decreased the levels of TNF-α, IFN-γ, IL-22, IL-17C, IL-1β and IL-4, and increased the level of 25HVD3; silencing VDR expression additionally downregulated the expression levels of NF-кB, p-NF-кB, IKK, p-IKK, STAT3, p-STAT3, STAT4 and p-STAT4, and upregulated the level of VDR in TNF-α-induced HaCaT cells. It was concluded that ‘psoriasis 1’ exerts inflammation-suppressive effects in psoriasis by suppressing the NF-кB and STAT signaling pathways.

Mechanism of hif-1α mediated hypoxia-induced permeability changes in bladder endothelial cells.

This study aimed to investigate the mechanism of hypoxia-inducible factor-1 alpha (HIF-1α) mediated hypoxia-induced permeability changes in bladder endothelial cells. Models of in vitro hypoxic cell culture of bladder cancer, bladder cancer cells with low HIF-1α expression and HIF-1α RNA interference (RNAi) expression vector were established. Western blot and reverse transcription polymerase chain reaction (RT-PCR) were used to detect the expression of HIF-1α and vascular endothelial growth factor (VEGF) in each group. Bladder cell permeability was determined. Results showed that protein and mRNA expression of HIF-1α and VEGF at 3 and 12 h of hypoxia were significantly higher than normal control (P<0.05), and peaked at 12 h. HIF-1α and VEGF expression in the hypoxic group and hypoxic+3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole (YC-1) group were significantly higher than normal control (P<0.05), while expression in the hypoxic+YC-1 group was significantly lower than the hypoxic group (P<0.05). Bladder cell permeability in the hypoxic and hypoxic+YC-1 group were significantly increased compared to normal control (P<0.05), while in the hypoxic+YC-1 group was significantly decreased compared to the hypoxic group (P<0.05). Most of the cells in the stably transfected HIF-1α RNAi expression vector pcDNA6.2-GW/EmGFP-miR-siHIF-1α expressed green fluorescence protein (GFP) under fluorescence microscope. pcDNA6.2-GW/EmGFP-miR-siHIF-1α could significantly inhibit HIF-1α gene expression (P<0.05). HIF-1α and VEGF expression in the hypoxic group and siHIF-1α hypoxic group were significantly higher than normal group (P<0.05), while expression in the siHIF-1α hypoxic group was significantly lower than the hypoxic group (P<0.05). Findings suggest that HIF-1α is an important factor in the increase of bladder cancer cell permeability.

Expression of histone deacetylase 1 gene in human lung cancer cell line A549 and the effect on the radiation sensitivity of nude mouse transplantation tumor

Objective To investigate the expression of histone deacetylase 1 (HDAC1) gene in human lung cancer cell line A549 and the effect on the radiation sensitivity of nude mouse transplantation tumor. Methods HDAC1 small interfering RNA (siRNA) and recombinant expression vector pcDNA3.1-HDAC1 were transfected with A549 lung cancer cells, respectively. Stable transfected cell lines were screened by G418 selection. reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting were used to detect the expression levels of HDAC1 mRNA and HDAC1 protein in the cell; cell proliferation was measured by cell counting kit-8 (CCK-8). Clone formation experiment was used to observe the effect of up-regulation and down-regulation of HDAC1 expression on the radiation sensitivity of A549 cells, cell apoptosis was detected by transferase-mediated dUTP nick-end labelling (TUNEL) method. Nude mice experiment of transplantable tumor was applied for in vivo detection of the influence of up-regulated and down-regulation of HDAC1 expression combined with X-ray irradiation in the inhibition of lung cancer growth. Results The expression of HDAC1 gene and protein were significantly lower to 1.86±0.09 and 2.41±0.10 (P=0.015, 0.006) in HDAC1 cell line A549, and the HDAC1 up-regulation group were significantly higher to 3.65±0.14 and 5.41±0.12 (P=0.012, 0.008); The radiation sensitivity of lung cancer A549 cells was increased in the down-regulation group of HDAC1 (P=0.025), and the radiation sensitivity of lung cancer A549 cells was decreased (P=0.009)in the HDAC1 up-regulation group. Down-regulation of HDAC1 inhibited the proliferation of A549 lung cancer, increased the rate of apoptosis to (42.17±3.19)% (P=0.008), proliferation of A549 cells was significantly enhanced in HDAC1 up-regulated group, associated with reduced percentage of apoptotic cells (9.11±1.46)% (P=0.006); Average volume in the nude mice of HDAC1 down-regulated group (547.37±89.68) mm3 were significantly smaller than the control group (1 238.44±212.36) mm3 (P=0.008); furthermore, mean volume of tumor was greater in the HDAC1 up-regulated group than in the control group (1 681.27±195.37) mm3 (P=0.006). After 20 Gy γ irradiation, the average volume of HDAC1 down-regulated group were significantly decreased (214.15±54.26) mm3 (P=0.011). Compared with that before irradiation, the average volume of the tumor in the nude mice was not significantly changed after irradiation (P=0.264). Conclusion Down-regulated expression of HDAC1 can inhibit the growth of human lung cancer A549 cells in nude mice, enhance the radiation sensitivity of tumor cells, whereas up-regulated expression of HDAC1 can produce tumor radiation resistance. Key words: Lung cancer; A549 cell; Histone deacetylase 1; Transplantable tumor; Nude mice

Silencing of histone deacetylase SlHDT3 delays fruit ripening and suppresses carotenoid accumulation in tomato

The acetylation levels of histones on lysine residues are regulated by histone acetyltransferases and histone deacetylases, which play an important but understudied role in the control of gene expression in plants. There is an increasing research focus on histone deacetylation in crops, but to date, there is little information regarding tomato. With the aim of characterizing the tomato HD2 family of histone deacetylases, an RNA interference (RNAi) expression vector of SlHDT3 was constructed and transformed into tomato plants. The time of fruit ripening was delayed and the shelf life of the fruit was prolonged in SlHDT3 RNAi lines. The accumulation of carotenoid was decreased by altering of the carotenoid pathway flux. Ethylene content was also reduced and expression of ethylene biosynthetic genes (ACS2, ACS4 and ACO1, ACO3) and ripening-associated genes (RIN, E4, E8, PG, Pti4 and LOXB) was significantly down-regulated in SlHDT3 RNAi lines. The expression of genes involved in fruit cell wall metabolism (HEX, MAN, TBG4, XTH5 and XYL) was inhibited compared with wild type. These results indicate that SlHDT3 functions as a positive regulator of fruit ripening by affecting ethylene synthesis and carotenoid accumulation and that SlHDT3 lies upstream of SlMADS-RIN in the fruit ripening regulatory network.

The tomato histone deacetylase SlHDA1 contributes to the repression of fruit ripening and carotenoid accumulation

Histone deacetylation is one of the well characterized post-translational modifications related to transcriptional repression in eukaryotes. The process of histone deacetylation is achieved by histone deacetylases (HDACs). Over the last decade, substantial advances in our understanding of the mechanism of fruit ripening have been achieved, but the role of HDACs in this process has not been elucidated. In our study, an RNA interference (RNAi) expression vector targeting SlHDA1 was constructed and transformed into tomato plants. Shorter fruit ripening time and decreased storability were observed in SlHDA1 RNAi lines. The accumulation of carotenoid was increased through an alteration of the carotenoid pathway flux. Ethylene content, ethylene biosynthesis genes (ACS2, ACS4 and ACO1, ACO3) and ripening-associated genes (RIN, E4, E8, Cnr, TAGL1, PG, Pti4 and LOXB) were significantly up-regulated in SlHDA1 RNAi lines. In addition, the expression of fruit cell wall metabolism genes (HEX, MAN, TBG4, XTH5 and XYL) was enhanced compared with wild type. Furthermore, SlHDA1 RNAi seedlings displayed shorter hypocotyls and were more sensitive to ACC (1-aminocyclopropane-1-carboxylate) than the wild type. The results of our study indicate that SlHDA1 functions as a negative regulator of fruit ripening by affecting ethylene synthesis and carotenoid accumulation.

Suppression of the MADS-box gene SlMBP8 accelerates fruit ripening of tomato (Solanum lycopersicum)

MADS-box genes encode important transcription factors that are involved in many biological processes of plants, including fruit ripening. In our research, a MADS-box gene, SlMBP8, was identified, and its tissue-specific expression profiles were analysed. SlMBP8 was highly expressed in fruits of the B+4 stage, in senescent leaves and in sepals. To further characterize its function, an RNA interference (RNAi) expression vector of SlMBP8 was constructed and transferred into tomato. In the transgenic plants, the ripening of fruits was shortened by 2–4 days compared to that of wild type. At the same time, carotenoids accumulated to higher levels and the expression of phytone synthase 1 (PSY1), phytoene desaturase (PDS) and ς-carotene desaturase (ZDS) was enhanced in RNAi fruits. The transgenic fruits and seedlings showed more ethylene production compared with that of the wild type. Furthermore, SlMBP8-silenced seedlings displayed shorter hypocotyls due to higher endogenous ethylene levels, suggesting that SlMBP8 may modulates the ethylene triple response negatively. A yeast two-hybrid assay indicated that SlMBP8 could interact with SlMADS-RIN. Besides, the expression of ethylene-related genes, including ACO1, ACO3, ACS2, ERF1, E4 and E8, was simultaneously up-regulated in transgenic plants. In addition, SlMBP8-silenced fruits showed higher ethylene production, suggesting that suppressed expression of SlMBP8 promotes carotenoid and ethylene biosynthesis. In addition, the fruits of transgenic plants displayed more rapid water loss and decreased storability compared to wild type, which was due to the significantly induced expressions of cell wall metabolism genes such as PG, EXP, HEX, TBG4, XTH5 and XYL. These results suggest that SlMBP8 plays an important role in fruit ripening and softening.

Lengthening of high-yield production levels of monoclonal antibody-producing Chinese hamster ovary cells by downregulation of breast cancer 1

The establishment process of high-producing Chinese hamster ovary (CHO) cells for therapeutic protein production is usually laborious and time consuming because of the low probability of obtaining stable, high-producing clones over a long term. Thus, development of an efficient approach is required to establish stable, high-producing cells. This study presents a novel method that can efficiently establish sustainably high-producing cell lines by acceleration of transgene amplification and suppression of transgene silencing. The effects of breast cancer 1 (BRCA1) downregulation on gene amplification efficiency and long-term productivity were investigated in CHO cells. Small interfering RNA expression vectors against BRCA1 were transfected into the CHO DG44-derived antibody-producing cell clone. Individual cell clones were obtained after induction of gene amplification in the presence of 400nM methotrexate, which were cultured until passage 20. BRCA1-downregulated cell clones CHO B1Sa and B1Sb displayed 2.2- and 1.6-fold higher specific production rates than the S-Mock clone. Fluorescence in situ hybridization showed that transgene amplification occurred at a high frequency in B1Sa and B1Sb clones. Moreover, B1Sa and B1Sb clones at 20 passages had approximately 3.5- and 5.3-fold higher productivity than the S-Mock clone. Histone modification analysis revealed a decrease in an active mark for transcription, trimethylation of histone H3 at lysine 4 (H3K4), in the transgene locus of the S-Mock clone. However, H3K4 trimethylation levels were not decreased in B1Sa and B1Sb clones during long term culture. Our results suggest that high-producing cells, which maintain their productivity long-term, were efficiently established by BRCA1 downregulation.

Agrobacterium-mediated transformation of the β-subunit gene in 7S globulin protein in soybean using RNAi technology.

The objective of this study was to use RNA interference (RNAi) to improve protein quality and decrease anti-nutritional effects in soybean. Agrobacterium tumefaciens-mediated transformation was conducted using RNAi and an expression vector containing the 7S globulin β-subunit gene. The BAR gene was used as the selective marker and cotyledonary nodes of soybean genotype Jinong 27 were chosen as explant material. Regenerated plants were detected by molecular biology techniques. Transformation of the β-subunit gene in the 7S protein was detected by PCR, Southern blot, and q-PCR. Positive plants (10 T0, and 6 T1, and 13 T2) were tested by PCR. Hybridization bands were detected by Southern blot analysis in two of the T1 transgenic plants. RNAi expression vectors containing the soybean 7S protein β-subunit gene were successfully integrated into the genome of transgenic plants. qRT-PCR analysis in soybean seeds showed a clear decrease in expression of the soybean β-subunit gene. The level of 7S protein β-subunit expression in transgenic plants decreased by 77.5% as compared to that of the wild-type plants. This study has established a basis for the application of RNAi to improve the anti-nutritional effects of soybean.

GW26-e2452 A Study in Construction of Short Hairpin Small Interfering RNA Expression Vector Target Lectin Like Oxidized Low Density Lipoprotein Receptor-1 Gene and Its Effect on foam cells

To construct the short hairpin small interfering RNA(shRNA) eukaryotic expression vector specific to mouse lectin like oxidized low density lipoprotein receptor 1(LOX-1) Gene and to observe its silencing effect on LOX-1 in RAW264.7 cells. (1) The pLOX-1-shRNA expression vector was constructed by

Promotion of Erythropoietic Differentiation in Hematopoietic Stem Cells by SOCS3 Knock-Down.

Suppressor of cytokine signaling 3 (SOCS3) plays an important role in mice fetal liver erythropoiesis, but the roles of SOCS3 in human hematopoietic stem cells (HSCs) have not been well investigated. In the present study, lentiviral small interference RNA expression vectors (shRNA) of SOCS3 were constructed and stably transferred into HSCs. We found that SOCS3 knockdown induced erythroid expansion in HSCs. Conversely, Ectopic expression of SOCS3 in progenitor cells blocked erythroid expansion and erythroid colony formation of HSCs. To further explore the involved mechanism, we compared gene expression profiles of SOCS3-shRNA tranduced HSCs with that of control HSCs by whole genome microarrays. The results indicated that cell developmental process related genes, especially hematopoietic lineage-specific genes, associated with the responses to SOCS3 in HSCs.Downexpression of SOCS3 in HSCs or differentiated erythroid progenitor cells induced a transcriptional program enriched for erythroid development relative genes. Our results proved that SOCS3 down-expression induced lineage commitment towards erythroid progenitor cell fate by activation of erythroid-specific gene in HSCs and provided new insight into the mechanism of erythropoietic development.

Suppression of wingless-type MMTV integration site family, member 1 expression by small interfering RNA inhibits U251 glioma cell growth in vitro.

A Wingless-type MMTV integration site family, member 1 (Wnt-1) RNA interference expression vector was constructed during the present study, which was used to transfect the glioma U251 cell line and investigate its effect on glioma. Two 21-base oligonucleotides complementary to the coding sequence that was flanking the loop sequence were designed to form a DNA hairpin template for the target small interfering RNA (siRNA). The siRNA templates were cloned into the siRNA expression vector, pGPU6/green fluorescent protein (GFP)/Neo and the sequence was confirmed by DNA sequencing. The pGPU6/GFP/Neo-short hairpin RNA (shRNA)-Wnt-1 vector was subsequently transfected into U251 cells, and reverse transcription polymerase chain reaction and western blot analysis were used to evaluate the Wnt-1 gene silencing effect on U251 cell growth by MTT assay and flow cytometry. The Wnt-1 protein expression was significantly reduced following transfection with the recombinant plasmid, as determined by western blot analysis of the transfected U251 cells. This transfection exhibited a significantly higher death rate, as shown by MTT. Thus, the present study demonstrated that the pGPU6/GFP/Neo-shRNA-Wnt-1 vector inhibited Wnt-1 protein expression. However, further investigations regarding the Wnt signaling pathway in glioma pathogenesis are required.

Synergic silencing of costimulatory molecules prevents cardiac allograft rejection

BackgroundWhile substantial progress has been made in blocking acute transplant rejection with the advent of immune suppressive drugs, chronic rejection, mediated primarily by recipient antigen presentation, remains a formidable problem in clinical transplantation. We hypothesized that blocking co-stimulatory pathways in the recipient by induction of RNA interference using small interference RNA (siRNA) expression vectors can prolong allogeneic heart graft survival.MethodVectors expressing siRNA specifically targeting CD40 and CD80 were prepared. Recipients (BALB/c mice) were treated with CD40 and/or CD80 siRNA expression vectors via hydrodynamic injection. Control groups were injected with a scrambled siRNA vector and sham treatment (PBS). After treatment, a fully MHC-mismatched (BALB/c to C57/BL6) heart transplantation was performed.ResultAllogeneic heart graft survival (>100 days) was approximately 70% in the mice treated simultaneously with CD40 and CD80 siRNA expression vectors with overall reduction in lymphocyte interstitium infiltration, vascular obstruction, and edema. Hearts transplanted into CD40 or CD80 siRNA vector-treated recipients had an increased graft survival time compared to negative control groups, but did not survive longer than 40 days. In contrast, allogenic hearts transplanted into recipients treated with scrambled siRNA vector and PBS stopped beating within 10–16 days. Real-time PCR (RT-PCR) and flow cytometric analysis showed an upregulation of FoxP3 expression in spleen lymphocytes and a concurrent downregulation of CD40 and CD80 expression in splenic dendritic cells of siRNA-treated mice. Functional suppressive activity of splenic dendritic cells (DCs) isolated from tolerant recipients was demonstrated in a mixed lymphocyte reaction (MLR). Furthermore, DCs isolated from CD40- and CD80-treated recipients promoted CD4 + CD25 + FoxP3+ regulatory T cell differentiation in vitro.ConclusionThis study demonstrates that the simultaneous silencing of CD40 and CD80 genes has synergistic effects in preventing allograft rejection, and may therefore have therapeutic potential in clinical transplantation.

The effect of checkpoint kinase 1 small interfering RNA on cisplatin induced esophageal carcinoma transplanted tumor cells biological behavior in vivo

Objective Constructing the nude mouse esophageal cancer transplanted tumor model,to observe the effect of checkpoint kinase 1 (Chk1) small interfering RNA (siRNA) interference expression vector on cisplatin induced esophageal carcinoma transplanted tumor cells biological behavior in vivo.Methods Group breeding tumor-burdened nude mouse,namely A groups:injection Sterile Saline; B group:injection Chk1 siRNA add cisplatin (5 mg/kg),C groups:injection cisplatin (5 mg/kg).Observing the transplantation tumor growth,using immunohistochemical to test the protein expression of Chk1 in the tumor tissues,using in situ hybridization to test the mRNA expression of Chk1 in the tumor tissues,using TdT-mediated dUTP nick end labeling (TUNEL) method to test the apoptosis situation of tumor tissues.Results The volume of transplanted tumor in the B group [(279.11 ± 27.23) mm3] was noticeably less than that in the C group [(512.41 ±35.18) mm3] and A group [(907.82 ±58.09) mm3],there were a statistical difference among three groups (P ＜ 0.01),the expression of Chk1 protein (26.87 ± 5.31) or mRNA (45.29 ± 6.47) in B group was significantly lower than that in the C group (protein:56.80 ± 7.00 ; mRNA:87.51 ± 8.38) and A group (protein:98.23 ± 15.67 ; mRNA:187.23 ± 18.55),there were a statistical difference among three groups (P ＜ 0.05),the resuhs of TUNEL method detection:the cells apoptosis index (AI) in group B [(21.03 ± 1.35) ％] is obviously higher than that in group C [(12.41 ± 1.23) ％] and group A [(1.34 ± 0.12) ％],there were a statistical difference among three groups (P ＜ 0.05).Conclusion Silencing Chk1 expression may strengthen the growth inhibition of nude mouse esophageal cancer transplantation tumor which cisplatin bring on,induce cell apoptosis of transplantation tumor. Key words: Esophageal squamous carcinoma; Checkpoint kinases 1 ; Cisplatin; Nud emice