FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2008383642> ?p ?o ?g. }

• W2008383642 endingPage "552" @default.
• W2008383642 startingPage "548" @default.
• W2008383642 abstract "BioTechniquesVol. 41, No. 5 BenchmarksOpen AccessDirect amplification of intron-containing hairpin RNA construct from genomic DNAYue-Hua Xiao, Meng-Hui Yin, Lei Hou & Yan PeiYue-Hua XiaoSouthwest University, Chongqing, P.R. ChinaSearch for more papers by this author, Meng-Hui YinSouthwest University, Chongqing, P.R. ChinaSearch for more papers by this author, Lei HouSouthwest University, Chongqing, P.R. ChinaSearch for more papers by this author & Yan Pei*Address correspondence to Yan Pei, Biotechnology Research Center, Southwest University, Chongqing 400716, P.R. China. e-mail: E-mail Address: peiyan3@swu.edu.cnSouthwest University, Chongqing, P.R. ChinaSearch for more papers by this authorPublished Online:21 May 2018https://doi.org/10.2144/000112295AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail RNA interference (RNAi) has been used to develop efficient strategies to silence targeted genes in a wide range of species (1–3). Hairpin RNA (hpRNA) constructs were commonly introduced into genomes to express hpRNAs that could induce degradation of target RNAs through RNAi machines (4,5). In plants, intron-containing hairpin RNA (ihpRNA) constructs with a spliceable intron as spacer sequence had the highest efficiency with 80% to approximately 100% transformants showing silencing of target genes (6–8). Currently, the ihpRNA technology has become one of the most powerful tools for gene discovery and gene engineering in plants (9–12).To facilitate generation of ihpRNA constructs, several generic vectors with a functional intron were reported (7,8). However, these methods generally required amplification of target sequences with long primers and/or several rounds of restrictions and ligations. Therefore, a simple and efficient method for rapid generation of ihpRNA constructs was urgently in demand. To this end, we developed a novel PCR-mediated method designated directed amplification of ihpRNA (DA-ihpRNA), to amplify an ihpRNA construct in one tube directly from genomic DNA. The resultant ihpRNA construct could be cloned into any expression vector as conveniently as a sense or antisense gene.The DA-ihpRNA method is depicted in Figure 1A. The final ihpRNA construct was designed to contain inverted repeats of an exon (Figure 1 A, exon2) with its flanking upstream intron as spacer sequence. Two primers, the flanking primer and the bridge primer, were used, and the concentration of the bridge primer was lowered to perform an asymmetric PCR. In the early cycles, a fragment consisting of the intron and exon2 was exponentially amplified. With the bridge primer exhausted in the late cycles, the main amplification would be the linear amplification of the antisense strand of the intron and exon2, which was primed by the flanking primer. The bridge primer contained a 5′ heel reverse complementary to the 5′ end of exon2, and the 3′ ends of the excessive antisense strands would anneal to the internal complementary sequences in the same or another single strands and initiate the synthesis of the reverse complementary sequence of exon2 (Figure 1 A). The resultant single strands would form the antisense strand of the final ihpRNA construct and could be used as a template to synthesize the double-stranded ihpRNA construct in the next PCR cycle.Figure 1. Direct amplification of intron-containing hairpin RNA (DA-ihpRNA) construct.(A) The DA-ihpRNA method. The genomic DNA is simplified as two exons intervened by an intron with the position indicated in the sense strand. The positions and directions of the bridge primers (BP) and the flanking primer (FP) are represented by arrows. Complementary sequences in the DNA strands (including primers, templates, and amplification products) are shown with bars of the same color. (B) Amplification of GhRacA ihpRNA construct from cloned genomic GhRacA gene. (C) Amplification of GhRacA ihpRNA construct from cotton genomic DNA. In panels B and C, lane M is DNA marker D200 (Dingguo, Beijing, China); lanes 1–4 are the amplification products with the bridge primers (BP) of 400, 200, 40, and 20 nM, respectively. The fragments consisting of intron 7 and exon 8 (I7+E8) and those containing inverted repeats of exon 8 and the intervening intron 7 (E8+I7+E8) are indicated, and the asterisks mark the putative single strands of intron 7 and exon 8. (D) The sequence of the amplified ihpRNA construct of GhRacA. The inverted repeats of GhRacA exon 8 are shaded in gray arrows. The positions of the exon and introns are indicated by black arrows. The sequences corresponding to the primers are underlined. Dots represent the internal sequences of exon 8 and intron 7. The sequence positions are indicated by the numbers.As a test, the DA-ihpRNA method was first used to amplify an ihpRNA construct of a small GTPase protein gene of cotton (GhRacA; GenBank® accession no. DQ667981) (13). The target ihpRNA construct consisted of two inverted repeats of the GhRacA exon 8 intervened by the flanking upstream intron (intron 7). The PCR of 25 µL contained 1 µL boiled Escherichia coli culture harboring the cloned genomic GhRacA gene or around 100 ng cotton genomic DNA, 1× Ex Taq™ buffer (TaKaRa, Dalian, China), 200 µM each dNTPs, 2 mM MgCl2, 400 nM flanking primer (Figure 1D, 5′-CGACTTGATCCTGATTGTCT-3′, annealing to the 3′ end of GhRacA exon 8), 1.5 U Ex Taq DNA polymerase (TaKaRa), and various concentrations (400, 200, 40, or 20 nM) of the bridge primer (Figure 1D, 5′-AAGTTCCTCCCGCAGAAGCCAAGTCGAGGATGTC-3′, the sequences annealed to the 3′ end of exon 7 are in bold, while those that are reverse complementary to the 5′ end of exon 8 are in italic). The reaction started at 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 56°C for 30 s, 72°C for 30 s, and a final extension at 72°C for 5 min. The interest fragment was gel-purified, cloned into a vector pUCm-T (Sangon, Shanghai, China) by AT cloning according to manufacturer′s instructions, and sequenced by Invitrogen (Shanghai, China) in an ABI PRISM® 3700 DNA sequence analyzer (Applied Biosystems, Foster City, CA, USA).As shown in Figure 1, B and C, similar amplification products were obtained with the cloned and uncloned genomic DNA as template. When the concentration of the bridge primer was high, the fragment containing a single copy of the intron and the target exon was the only amplification product. However, when the bridge primer concentration was lowered to 40 and 20 nM (one-tenth and one-twentieth of the concentration of the flanking primer, respectively), a fragment of about 700 bp was amplified. Sequencing analysis confirmed that this fragment was 689 bp in length and contained two self-complementary arms and a spacer as expected (Figure 1D), suggesting that the ihpRNA construct of the GhRacA gene was successfully amplified from both cloned and uncloned cotton genomic DNA. Additionally, we had harnessed the DA-ihpRNA method to amplify a total of 14 ihpRNA constructs, with the inverted arms of 102–352 bp and the spacer of 85–693 bp, from cotton, rice, and Arabidopsis (data not shown). With appropriate primers, all these ihpRNA constructs (100%) had been successfully amplified and cloned, indicating that the DA-ihpRNA method was quite reliable.High efficiency of DA-ihpRNA method required the template DNA containing an intron-containing spacer of appropriate length (100 to approximately 500 bp), which presumably facilitated annealing of the 3′ end of excess single strands to internal complementary sequence. Furthermore, to decrease the PCR suppression effect of the inverted repeats in the last step (14), the sequence of inverted arm should excess 100 bp. However, compared with the previous methods to generate ihpRNA constructs (7,8), the DA-ihpRNA method bypassed the construction of generic vectors, required less or shorter primers, and reduced cloning steps. Most important of all, by using this method, ihpRNA constructs could be amplified directly from uncloned genomic DNAs as long as the sequence was available. With several genomes sequenced and more and more sequence information available in the web, the DA-ihpRNA method would be increasingly valuable to generate ihpRNA constructs for the functional analyses of various intron-containing genes.In addition to the ihpRNA constructs, other hpRNA constructs without intron in the spacer were also useful in some systems, showing high gene silencing efficiency (2,3). In these cases, any DNA of appropriate length could be used as a template to amplify hpRNA construct using the DA-ihpRNA method, which might broaden the application of the DA-ihpRNA method to generate constructs for RNAi. In short, with suitable template available, the DA-ihpRNA method was simple and efficient for rapid generation of hpRNA constructs for gene functional analyses via RNAi.AcknowledgmentsThis work was partially supported by the China National Basic Research Program (973) (2004CB117300 to L.H.) and by the National Natural Science Foundation of China (30200177 and 30471055 to Y.-H.X.).Competing Interests StatementThe authors declare no competing interests.References1. Dykxhoorn, D.M., C.D. Novina, and P.A. Sharp. 2003. Killing the messenger: short RNAs that silence gene expression. Nat. Rev. Mol. Cell Biol. 4:457–467.Crossref, Medline, CAS, Google Scholar2. McGinnis, K., V. Chandler, K. Cone, H. Kaeppler, S. Kaeppler, A. Kerschen, C. Pikaard, E. Richards, et al.. 2005. Transgene-induced RNA interference as a tool for plant functional genomics. Methods Enzymol. 392:1–24.Crossref, Medline, CAS, Google Scholar3. Heilersig, B.H.J.B., A.E.H.M. Loonen, A.M.A. Wolters, and R.G.F. Visser. 2006. Presence of an intron in inverted repeat constructs does not necessarily have an effect on efficiency of post-transcriptional gene silencing. Mol. Breed. 17:307–316.Crossref, CAS, Google Scholar4. Lu, S., R. Shi, C.C. Tsao, X. Yi, L. Li, and V.L. Chiang. 2004. RNA silencing in plants by the expression of siRNA duplexes. Nucleic Acids Res. 21:e171.Crossref, Google Scholar5. Tomari, Y. and P.D. Zamore. 2005. Perspective: machines for RNAi. Genes Dev. 19:517–529.Crossref, Medline, CAS, Google Scholar6. Smith, N.A., S.P. Singh, M.B. Wang, P.A. Stoutjesdijk, A.G. Green, and P.M. Waterhouse. 2000. Total silencing by intron-spliced hairpin RNAs. Nature 407:319–320.Crossref, Medline, CAS, Google Scholar7. Wesley, S.V., C.A. Helliwell, N.A. Smith, M. Wang, D.T. Rouse, Q. Liu, P.S. Gooding, S.P. Singh, et al.. 2001. Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J. 27:581–590.Crossref, Medline, CAS, Google Scholar8. Miki, D. and K. Shimamoto. 2004. Simple RNAi vectors for stable and transient suppression of gene function in rice. Plant Cell Physiol. 45:490–495.Crossref, Medline, CAS, Google Scholar9. Wang, M.B. and P.M. Waterhouse. 2001. Application of gene silencing in plants. Curr. Opin. Plant Biol. 5:146–150.Crossref, Google Scholar10. Miki, D., R. Itoh, and K. Shimamoto. 2005. RNA silencing of single and multiple members in a gene family of rice. Plant Physiol. 138:1903–1913.Crossref, Medline, CAS, Google Scholar11. Liu, Q., S.P. Singh, and A.G. Green. 2002. High-stearic and high-oleic cottonseed oils produced by hairpin RNA-mediated post-transcriptional gene silencing. Plant Physiol. 129:1732–1743.Crossref, Medline, CAS, Google Scholar12. Andersson, M., M. Melander, P. Pojmark, H. Larsson, L. Bülow, and P. Hofvander. 2005. Targeted gene suppression by RNA interference: an efficient method for production of high-amylose potato lines. J. Biotechnol. 123:137–148.Crossref, Google Scholar13. Li, X.B., Y.H. Xiao, M. Luo, L. Hou, D.M. Li, X.Y. Luo, and Y. Pei. 2005. Cloning and expression analysis of two Rac genes from cotton (Gossypium hirsutum L.). Yi Chuan XueBao 32:72–78.Medline, Google Scholar14. Lukyyanov, K.A., N.G. Gurskaya, E.A. Bogdanova, and S.A. Lukyanov. 1999. Selective suppression of polymerase chain reaction. Russ. J. Bioorganic Chem. 25:141–147.Google ScholarFiguresReferencesRelatedDetailsCited ByAuxin Regulates Cotton Fiber Initiation via GhPIN-Mediated Auxin Transport29 December 2016 | Plant and Cell Physiology, Vol. 223Artificial microRNA mediated gene silencing in plants: progress and perspectives15 July 2014 | Plant Molecular Biology, Vol. 86, No. 1-2Gibberellin Overproduction Promotes Sucrose Synthase Expression and Secondary Cell Wall Deposition in Cotton Fibers9 May 2014 | PLoS ONE, Vol. 9, No. 5One-step cloning of intron-containing hairpin RNA constructs for RNA interference via isothermal in vitro recombination system17 May 2013 | Planta, Vol. 238, No. 2Molecular Cloning and Characterization of a Cytokinin Dehydrogenase Gene from Upland Cotton (Gossypium hirsutum L.)15 April 2011 | Plant Molecular Biology Reporter, Vol. 30, No. 1Gibberellin 20-oxidase promotes initiation and elongation of cotton fibers by regulating gibberellin synthesisJournal of Plant Physiology, Vol. 167, No. 10One‐step, zero‐background ligation‐independent cloning intron‐containing hairpin RNA constructs for RNAi in plants12 April 2010 | New Phytologist, Vol. 187, No. 1Rapid one-step construction of hairpin RNABiochemical and Biophysical Research Communications, Vol. 383, No. 4A Novel Approach Obtaining Intron-Containing Hairpin RNA Constructs22 May 2014 | Bioscience, Biotechnology, and Biochemistry, Vol. 72, No. 2 Vol. 41, No. 5 STAY CONNECTED Metrics History Received 10 July 2006 Accepted 22 August 2006 Published online 21 May 2018 Published in print November 2006 Information© 2006 Author(s)AcknowledgmentsThis work was partially supported by the China National Basic Research Program (973) (2004CB117300 to L.H.) and by the National Natural Science Foundation of China (30200177 and 30471055 to Y.-H.X.).Competing Interests StatementThe authors declare no competing interests.PDF download" @default.
• W2008383642 created "2016-06-24" @default.
• W2008383642 creator A5013890493 @default.
• W2008383642 creator A5040599820 @default.
• W2008383642 creator A5040806189 @default.
• W2008383642 creator A5074656318 @default.
• W2008383642 date "2006-11-01" @default.
• W2008383642 modified "2023-10-18" @default.
• W2008383642 title "Direct amplification of intron-containing hairpin RNA construct from genomic DNA" @default.
• W2008383642 cites W1496492558 @default.
• W2008383642 cites W2010791234 @default.
• W2008383642 cites W2011271253 @default.
• W2008383642 cites W2085681314 @default.
• W2008383642 cites W2106789843 @default.
• W2008383642 cites W2128946743 @default.
• W2008383642 cites W2135874498 @default.
• W2008383642 cites W2136861504 @default.
• W2008383642 cites W2143889640 @default.
• W2008383642 cites W2150816271 @default.
• W2008383642 cites W2171757104 @default.
• W2008383642 cites W2354977637 @default.
• W2008383642 doi "https://doi.org/10.2144/000112295" @default.
• W2008383642 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/17140110" @default.
• W2008383642 hasPublicationYear "2006" @default.
• W2008383642 type Work @default.
• W2008383642 sameAs 2008383642 @default.
• W2008383642 citedByCount "12" @default.
• W2008383642 countsByYear W20083836422012 @default.
• W2008383642 countsByYear W20083836422013 @default.
• W2008383642 countsByYear W20083836422014 @default.
• W2008383642 countsByYear W20083836422016 @default.
• W2008383642 countsByYear W20083836422019 @default.
• W2008383642 crossrefType "journal-article" @default.
• W2008383642 hasAuthorship W2008383642A5013890493 @default.
• W2008383642 hasAuthorship W2008383642A5040599820 @default.
• W2008383642 hasAuthorship W2008383642A5040806189 @default.
• W2008383642 hasAuthorship W2008383642A5074656318 @default.
• W2008383642 hasBestOaLocation W20083836421 @default.
• W2008383642 hasConcept C104317684 @default.
• W2008383642 hasConcept C153911025 @default.
• W2008383642 hasConcept C17757408 @default.
• W2008383642 hasConcept C54355233 @default.
• W2008383642 hasConcept C552990157 @default.
• W2008383642 hasConcept C67705224 @default.
• W2008383642 hasConcept C70721500 @default.
• W2008383642 hasConcept C86803240 @default.
• W2008383642 hasConcept C94671646 @default.
• W2008383642 hasConceptScore W2008383642C104317684 @default.
• W2008383642 hasConceptScore W2008383642C153911025 @default.
• W2008383642 hasConceptScore W2008383642C17757408 @default.
• W2008383642 hasConceptScore W2008383642C54355233 @default.
• W2008383642 hasConceptScore W2008383642C552990157 @default.
• W2008383642 hasConceptScore W2008383642C67705224 @default.
• W2008383642 hasConceptScore W2008383642C70721500 @default.
• W2008383642 hasConceptScore W2008383642C86803240 @default.
• W2008383642 hasConceptScore W2008383642C94671646 @default.
• W2008383642 hasIssue "5" @default.
• W2008383642 hasLocation W20083836421 @default.
• W2008383642 hasLocation W20083836422 @default.
• W2008383642 hasLocation W20083836423 @default.
• W2008383642 hasOpenAccess W2008383642 @default.
• W2008383642 hasPrimaryLocation W20083836421 @default.
• W2008383642 hasRelatedWork W1976736675 @default.
• W2008383642 hasRelatedWork W2039352533 @default.
• W2008383642 hasRelatedWork W2041335525 @default.
• W2008383642 hasRelatedWork W2072834586 @default.
• W2008383642 hasRelatedWork W2081600652 @default.
• W2008383642 hasRelatedWork W2092554027 @default.
• W2008383642 hasRelatedWork W2109717569 @default.
• W2008383642 hasRelatedWork W2334012009 @default.
• W2008383642 hasRelatedWork W2376186192 @default.
• W2008383642 hasRelatedWork W4242521457 @default.
• W2008383642 hasVolume "41" @default.
• W2008383642 isParatext "false" @default.
• W2008383642 isRetracted "false" @default.
• W2008383642 magId "2008383642" @default.
• W2008383642 workType "article" @default.