Construction and Evaluation of Short Hairpin RNAs for Knockdown of Metadherin mRNA

Background: Short hairpin RNA (shRNA) has proven to be a powerful tool to study genes’ function through RNA interference mechanism. Three different methods have been used in previous studies to produce shRNA expression vectors including oligonucleotide-based cloning, polymerase chain reaction (PCR)-based cloning, and primer extension PCR approaches. The aim of this study was designing a reliable and simple method according to the primer extension strategy for constructing four shRNA vectors in order to target different regions of Metadherin (MTDH) mRNA in human leukemic cell line Jurkat. Methods: Oligonucleotides for construction of four shRNA vectors were designed, synthesized and fused to U6 promoter. Each U6-shRNA cassette was cloned into a pGFP-V-RS vector. MTDH shRNAs were transfected into the Jurkat cell line by using the electroporation method. The ability of shRNAs to knock down MTDH mRNA was analyzed through qRT-PCR. Apoptosis assay was used to evaluate the effect of down regulation of MTDH expression on cell integrity. Results: A significant reduction (about 80%) in the expression levels of MTDH mRNA and an increase in the percentages of apoptotic cells (about 20%) were observed in the test group in comparison with control. Conclusion: MTDH shRNA constructs effectively inhibited gene expression. However, simplicity and inexpensiveness of the method were additional advantages for its application.

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4 CHARACTERIZATION OF LENTIVIRAL SHORT-HAIRPIN RNA EXPRESSION VECTORS CONTAINING SINGLE OR MULTIPLE BOVINE POLYMERASE III PROMOTERS

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab4 ◽

2010 ◽

Vol 22 (1) ◽

pp. 160

Author(s):

M. Peoples ◽

M. Westhusin ◽

M. Golding ◽

C. Long

Keyword(s):

Luciferase Activity ◽

Lentiviral Vector ◽

Lentiviral Vectors ◽

Expression Vectors ◽

Luciferase Reporter ◽

Second Phase ◽

Shrna Expression ◽

Significant Difference ◽

Short Hairpin ◽

Pol Iii

Lentiviral vectors have become an important and efficient molecular biology tool to integrate foreign DNA into target genomes. These vectors have been previously used in our laboratory to make cloned transgenic fetuses expressing short-hairpin RNAs (shRNAs) targeting the caprine prion mRNA (Golding et al. 2006 Proc. Natl. Acad. Sci. USA 103, 5285-5290) and bovine myostatin mRNA. Specially designed shRNAs have a robust ability to decrease protein expression by initiating a mRNA destruction pathway or by translational inhibition. However, initial experiments targeting foot and mouth (FMDV) viral RNA have indicated that polymerase (Pol) II promoters may be unable to produce enough mature shRNA particles to significantly knock down viral replication in vitro. The goal of this research project was to identify and utilize bovine Pol III promoters to express shRNAs in lentiviral vectors and to express multiple unique shRNAs from a single lentiviral vector using different Pol III promoters. This goal is particularly important to the successful reduction of FMDV replication in a cell, as it limits random mutations from escaping the shRNA-mediated viral genome destruction. The 3 bovine Pol III promoters we selected were 7sk, U6-2, and H1. They were individually amplified from the same genomic DNA preparation. The promoters were inserted immediately upstream of our shRNA expression sequence, resulting in lentiviral vectors designated GT-b7sk, GT-bU6-2, and GT-bH1.To confirm that the promoters were functional, a luciferase reporter assay was performed in HEK 293T cells, where each vector expressed either a shRNA targeting luciferase (luc) or a non-specific shRNA.All promoters expressing luc shRNA resulted in significant reduction of luciferase activity between 68 and 80% compared with non-targeting controls. In addition, there was no significant difference between Pol III promoters when analyzing reduced luciferase activity. In the second phase of the study, we developed 7 unique combinations of 2 or 3 Pol III shRNA expression cassettes to test individual shRNA function with one shRNA designed to target luciferase and the others non-targeting. In multiple Pol III expression constructs, the U6-2 and 7sk promoters resulted in the greatest reduction of luciferase activity at 89 and 95%, respectively. In addition, luciferase activity was reduced to the greatest extent when the luc shRNA was expressed from the second (82%) or third (87%) Pol III cassette. Overall, bovine Pol III-based promoters are effective at expressing shRNAs from a lentiviral vector. In addition, multiple Pol III shRNA expression cassettes can be inserted into a single lentiviral vector and still achieve significant reduction of target protein. These vectors will be used to create transgenic cattle and pigs that express multiple shRNAs targeting the FMDV genome with hopes of creating animals that are resistant to FMDV.

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Primer extension-based method for the generation of a siRNA/miRNA expression vector

Physiological Genomics ◽

10.1152/physiolgenomics.00005.2007 ◽

2007 ◽

Vol 31 (3) ◽

pp. 554-562 ◽

Cited By ~ 24

Author(s):

Deming Gou ◽

Honghao Zhang ◽

Pradyumna S. Baviskar ◽

Lin Liu

Keyword(s):

High Throughput Screening ◽

Mammalian Cells ◽

Promoter Activity ◽

Expression Vector ◽

A549 Cells ◽

Primer Extension ◽

Expression Vectors ◽

Shrna Expression ◽

Survivin Promoter ◽

Shrna Expression Vector

RNA interference (RNAi) has become a powerful technique for studying gene function, biological pathways, and the physiology of diseases. Typically, the RNAi response in mammalian cells is mediated by small interfering RNA (siRNA). The use of synthesized siRNA to silence gene is relatively quick and easy, but it is costly with transient effects. A short hairpin RNA (shRNA) with complementary sense and antisense sequences of a target gene separated by a loop structure results in gene silencing that is as effective as chemically synthesized siRNA with fewer limitations. However, current methods for constructing shRNA vectors require the synthesis of long oligonucleotides, which is costly and often suffers from mutation problems during synthesis. Here, we report an alternative approach to generate a shRNA expression vector with high efficacy. We utilized shorter (≤50-nucleotide) primers to generate a shRNA insert by the primer extension method. Our new approach for the construction of shRNA expression vectors dramatically reduced the possibility of mutations. Using this method, we constructed a microRNA (miRNA) library, which facilitates the expression of 254 matured miRNAs. We also performed high-throughput screening of miRNAs involved in the regulation of human Survivin promoter activity in lung A549 cells. We found that the expression of miR-192, 199a, 19a, 20a, 213, and 371 caused the activation of the Survivin promoter whereas miR-302b*, 34a, 98, 381, 463 and 471 decreased the Survivin promoter activity.

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