Cloning and expression analysis of U6 promoters in Panax quinquefolius / 中国中药杂志

The U6 promoter is an important element driving sgRNA transcription in the CRISPR/Cas9 system. Seven PqU6 promo-ter sequences were cloned from the gDNA of Panax quinquefolium, and the transcriptional activation ability of the seven promoters was studied. In this study, seven PqU6 promoter sequences with a length of about 1 300 bp were cloned from the adventitious roots of P. quinquefolium cultivated for 5 weeks. Bioinformatics tools were used to analyze the sequence characteristics of PqU6 promoters, and the fusion expression vectors of GUS gene driven by PqU6-P were constructed. Tobacco leaves were transformed by Agrobacterium tumefaciens-mediated method for activity detection. The seven PqU6 promoters were truncated from the 5'-end to reach 283, 287, 279, 289, 295, 289, and 283 bp, respectively. The vectors for detection of promoter activity were constructed with GUS as a reported gene and used to transform P. quinquefolium callus and tobacco leaves. The results showed that seven PqU6 promoter sequences(PqU6-1P to PqU6-7P) were cloned from the gDNA of P. quinquefolium, with the length ranged from 1 246 bp to 1 308 bp. Sequence comparison results showed that the seven PqU6 promoter sequences and the AtU6-P promoter all had USE and TATA boxes, which are essential elements affecting the transcriptional activity of the U6 promoter. The results of GUS staining and enzyme activity test showed that all the seven PqU6 promoters had transcriptional activity. The PqU6-7P with a length of 1 269 bp had the highest transcriptional activity, 1.31 times that of the positive control P-35S. When the seven PqU6 promoters were truncated from the 5'-end(PqU6-1PA to PqU6-7PA), their transcriptional activities were different in tobacco leaves and P. quinquefolium callus. The transcriptional activity of PqU6-7PA promoter(283 bp) was 1.59 times that of AtU6-P promoter(292 bp) when the recipient material was P. quinquefolium callus. The findings provide more ideal endogenous U6 promoters for CRISPR/Cas9 technology in ginseng and other medicinal plants.

An update and perspectives on the use of promoters in plant genetic engineering

Genetically engineered plants have varied applications in agriculture for enhancing the values of food and feed.Genetic engineering aims to introduce selected genetic regions with desirable traits into target plants for bothspatial and temporal expressions. Promoters are the key elements responsible for regulating gene expressionsby modulating the transcription factors (TFs) through recognition of RNA polymerases. Based on theirrecognition and expression, RNA polymerases were categorized into RNA pol II and pol III promoters.Promoter activity and specificity are the two prime parameters in regulating the transgene expression. Since theuse of constitutive promoters like Cauliflower mosaic virus (CaMV) 35S may lead to adverse effects on nontarget organisms or ecosystem, inducible/tissue specific promoters and/or the RNA pol III promoters providemyriad opportunities for gene expressions with controlled regulation and with minimum adverse effects.Besides their role in transgene expression, their influence in synthetic biology and genome editing are alsodiscussed. This review provides an update on the importance, current prospects, and insight into the advantagesand disadvantages of promoters reported thus far would help to utilize them in the endeavour to developnutritionally and agronomically improved transgenic crops for commercialization.

Comparison between Let-7a and U6 as an internal reference for RT-qPCR of miRNAs in rat cartilage / 西安交通大学学报(医学版)

Objective To evaluate the stability of U6 and let-7a as internal reference genes of miRNAs in RTqPCR by using femoral head samples of cartilage tissue from inbred DA rats.Methods Total RNA was extracted from femoral head cartilage tissues of female DA rats at three different time points,i.e.at birth (D0),ablactation (D21) and maturation (D42).The expressions of different miRNAs (miR-1,-25,-26a,-140,-146a,-150,-181a,-195,-223 and-337) were detected by RT-qPCR using U6 or let-7a as the internal reference.The two sets of miR expression were compared with the results from Solexa sequencing in our pioneer work to evaluate the stability of the two internal references.Results The relative values of U6 (P =0.045) and let-7a (P =0.021 5) revealed significant difference in the D42 sample.Both in U6 and let-7a systems,miR-26a,-140,-223,and-337 showed a similar tendency in expression and quantification but miR-1 and-146a did not have significant differences.miR-25,-150,-181a and-195 differed significantly (P＜0.05).Comparison of absolute quantification results between the two generations' sequencing showed that let-7a is more stable than U6.Conclusion Let-7a is more suitable to be used as the internal reference gene in RT-qPCR for miRNAs in cartilage tissue.

U6 is unsuitable for normalization of serum miRNA levels in patients with sepsis or liver fibrosis

MicroRNA (miRNA) levels in serum have recently emerged as potential novel biomarkers for various diseases. miRNAs are routinely measured by standard quantitative real-time PCR (qPCR); however, the high sensitivity of qPCR demands appropriate normalization to correct for nonbiological variation. Presently, RNU6B (U6) is used for data normalization of circulating miRNAs in many studies. However, it was suggested that serum levels of U6 themselves might differ between individuals. Therefore, no consensus has been reached on the best normalization strategy in 'circulating miRNA'. We analyzed U6 levels as well as levels of spiked-in SV40-RNA in sera of 44 healthy volunteers, 203 intensive care unit patients and 64 patients with liver fibrosis. Levels of U6 demonstrated a high variability in sera of healthy donors, patients with critical illness and liver fibrosis. This high variability could also be confirmed in sera of mice after the cecal ligation and puncture procedure. Most importantly, levels of circulating U6 were significantly upregulated in sera of patients with critical illness and sepsis compared with controls and correlated with established markers of inflammation. In patients with liver fibrosis, U6 levels were significantly downregulated. In contrast, levels of spiked-in SV40 displayed a significantly higher stability both in human cohorts (healthy, critical illness, liver fibrosis) and in mice. Thus, we conclude that U6 levels in the serum are dysregulated in a disease-specific manner. Therefore, U6 should not be used for data normalization of circulating miRNAs in inflammatory diseases and previous studies using this approach should be interpreted with caution. Further studies are warranted to identify specific regulatory processes of U6 levels in sepsis and liver fibrosis.

Pre-mRNA splicing and retinitis pigmentosa / 中华实验眼科杂志

Retinitis pigmentosa (RP) is a large group of common hereditary eye diseases with highlyheterogeneous genetic background. Over forty genes with diverse functionalities are associated with RP and they include a set of ubiquitously expressed genes. These include five genes involved in the precursor messenger RNA( premRNA) splicing. Recent progress in disease gene identification for RP has established the involvement of pre-mRNA splicing as one important mechanism in the disease etiology and has shed light on the splicing process itself, a fundamental biological process. To this date, studies in this field have been focused on two major issues. First, how do the mutations of the adRP associated splicing factors (adRP-SF) affect the splicing function? Second, how do the mutations in these ubiquitously expressed genes lead to specific retinopathy? The two topics fit with the two continuous important steps of the disease pathogenesis. Recently, researchers have made a dramatic progress in the first topic. The identification of the SNRNP200 gene,the fifth adRP-SF and its relevant functional study has shown significance to the progress in the study of RP. Numerous investigations are also being carried out in addressing the second issue.Generation of a variety of models led to a better description of the pathological process of the disease. However, in respect to the key pathogenic mechanism,researchers are still puzzled with a number of confusing questions. In this commentary,the results from the latest investigations were summarized, and in particular,the difficulties in studying the molecular mechanism by which the pre-mRNA splicing deficiency causes RP were detailed.

Establishment and identification of siRNA eukaryotic expression vectors of recombinant targeting gene glucocorticoid receptor / 重庆医科大学学报

Objective:Establish eukaryotic expression vector of glucocorticoid receptor GR,to explore the relation of GR and tradition Chinese medicine.Methods:Recombinant were designed and established Supported by the National Natural Science Foundation of China(3047119)by targeting gene GR and plasmid pBS/U6-Neo based on GR mRNA sequence.Two pairs of oligonucleotides were synthesized and inserted into plasmid pBS/U6-Neo to generate siRNA eukaryotic expression vectors.DH5a strains were transformed.plasmids were extracted,and the recombinant sequences were identified by PCR and sequencing.Results:The result of recombinant sequence was the same as aim sequence.The recombinant vectors were established successfully.Conclusion:siRNA recombinant can be established successfully by RNAi technique,to use in further gene stability transfer and cultivate transgenic mouse.

A strategy for rapidly constructing the single and dual-site targeting plasmid-based RNAi vector / 基础医学与临床

Objective To develop a rapid method for construction of RNAi vector.Methods After inversely inserting the U6 and H1 polymerase Ⅲ promoters into the pBluescriptⅡ backbone vector,only with two short reverse complementary oligo nucleotides,the RNAi vector with one interference cassette could be constructed.Using two isoaudamers MunⅠ and EcoRⅠ with sticky ends,several cassettes could be fused together to form the ultimate multiple-site targeting RNAi vector.Using this method,we constructed RNAi vectors targeting green fluorescent protein(GFP) and firefly luciferase(LUC) gene separately or both for the test of knock down property of these vectors.Results Constructed single and two site targeting RNAi vectors got desirable knockdown effects,in which single site targeting vector could get about 90 percent knock down efficiency whereas two site targeting vector reached about 87.5 percent knock down efficiency either.Conclusion Our RNAi vector construction strategy is efficient and time-saving so that it can be used for knocking down several genes simultaneously in the same cell.

Construction of an expression vector directed by human U_6 small nuclear RNA promoter and identification of expression in gastric carcinoma cell / 西安交通大学学报(医学版)

Objective To construct an expression vector directed by hU_(6)snRNA promoter for synthesizing small RNA and to identify its functional activity in the gastric carcinoma cells——SGC-7901.Methods Using human genomic DNA as template,U_(6) snRNA promoter was obtained by PCR method,and then cloned into PUC19 vector to produce the recombinant plasmid PUC-hU_6-extra,which was sequenced and then transfected into gastric carcinoma cell——SGC-7901 with liposome.The effect of expression directed by U_6 promoter was detected by RT-PCR method,and the cell proliferation curve analysis was performed by stained dye.Results The hU_6 snRNA promoter with the first 27 nucleotides followed were successfully cloned into PUC19 plasmid.The recombinant vector could efficiently transcribe small RNA molecules and exerted no effect on cell proliferation in SGC-7901 cells in vitro.Conclusion We have successfully constructed the recombinant PUC-hU_6-extra plasmid vector that can efficiently transcribe small RNA molecules directed by hU_6 snRNA promoter in the gastric carcinoma cells——SGC-7901.

Computer-assisted design of TIMP-1 ribozyme / 第二军医大学学报

Objective: To design the ribozymes to cleave human TIMP 1 mRNA, and embed them into U 6snRNA to make them stable. Methods: Ribozymes were designed according to the “hammerhead structure” described by Symons.Computer was used to analyze the possible cleavage sites. Results: Three ribozymes targeting the nt123, nt299 and nt353 on TIMP 1 mRNA were designed. Embedding ribozyme in U 6snRNA had little effect on its binding with the substrate. Conclusion: Computer assisted design is indispensable in studying ribozyme. Embedding ribozymes in U 6snRNA may be a good way to solve the problems existing in ribozyme study. [