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Formation of Minimised Hairpin Template-transcribing Dumbbell Vectors for Small RNA Expression
用于小分子RNA表达的最小化发夹模板转录哑铃形载体生成   

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Abstract

A major barrier for using non-viral vectors for gene therapy is the short duration of transgene expression in postmitotic tissues. Previous studies showed transgene expression from conventional plasmid fell to sub-therapeutic level shortly after delivery even though the vector DNA was retained, suggesting transcription was silenced in vivo (Nicol et al., 2002; Chen et al., 2004). Emerging evidence indicates that plasmid bacterial backbone sequences are responsible for the transcriptional repression and this process is independent of CpG methylation (Chen et al., 2008). Dumbbell-shaped DNA vectors consisting solely of essential elements for transgene expression have been developed to circumvent these drawbacks. This novel non-viral vector has been shown to improve transgene expression in vitro and in vivo (Schakowski et al., 2001 and 2007). Here we describe a novel method for fast and efficient production of minimised small RNA-expressing dumbbell vectors. In brief, the PCR-amplified promoter sequence is ligated to a chemically synthesized hairpin RNA coding DNA template to form the covalently closed dumbbell vector. This new technique may facilitate applications of dumbbell-shaped vectors for preclinical investigation and human gene therapy.

Keywords: Dumbbell vector(哑铃形载体), Minimal vector(最小载体), Small RNA expression(小分子RNA表达), MiRNA(miRNA), shRNA(shRNA), Genetic therapy(基因疗法)

Background

With regard to delivery, a small vector size is advantageous improving extracellular transport including extravasation and diffusion through the extracellular matrix network as well as cellular uptake and nuclear diffusion. Various methods for dumbbell vector production have been developed over the time including methods for the generation of dumbbells expressing small RNAs such as small hairpin RNAs (shRNAs) and microRNAs (miRNAs) (Schakowski et al., 2001; Taki et al. 2004). These vectors usually harbour redundant sequences as the expressed RNAs are self-complementary. We eliminated redundant sequences generating minimised dumbbell vectors in which transcription goes around the hairpin structure of the dumbbell itself (Jiang et al., 2016). Such minimised dumbbell vectors can be as short as 130 bp representing the smallest expression vectors ever reported. An illustrated comparison between a conventional plasmid, a dumbbell harbouring a linear expression cassette, and a novel hairpin template-transcribing dumbbell vector is shown in Figure 1. This novel protocol facilitates the production of the new minimised small RNA expression dumbbell vectors.

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Figure 1. Structures of small hairpin RNA-expressing plasmid and dumbbell vectors. Upper two: conventional plasmid p-iPR-linear-s/as and dumbbell db-iPR-linear-s/as vectors with linear shRNA expression cassettes and integrated promoter-restriction endonuclease site element (iPR). Lower vector: minimized hairpin template (hp) dumbbell harboring an iPRT element. R indicates a restriction overhang ligation site. T indicates termination signal. IT indicates inverted termination signal. Loops L1 and L2 are (T)4 tetra loops.

Materials and Reagents

  1. 0.2-10 μl pipette tips, Corning® Isotip® filtered (Corning, catalog number: 4807 )
  2. 1-200 μl pipette tips (Corning, Axyge®, catalog number: TF-200-R-S )
  3. 100-1,000 μl pipette tips (Corning, Axygen®, catalog number: TF-1000-R-S )
  4. 1.5 ml microcentrifuge tubes (RNase, DNase and Pyrogen-Free) (Corning, Axygen®, catalog number: MCT-150-C )
  5. 0.2 ml thin-walled PCR tubes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3412 )
  6. Falcon® 50 ml conical centrifuge tubes (Corning, Falcon®, catalog number: 352070 )
  7. pSuper-basic vector (Oligoengine, catalog number: VEC-pBS-0002 )
  8. miRNA-coding oligonucleotide (PAGE purified) 5’-pGATCTAGCACGACTCGCAGCTCCCAAGAGCCTAACCCGTGGATTTAAACGGTAAACATCACAAGTTAGGGTCTCAGGGACTGAGAGGAGCGCAA-3’
  9. Oligonucleotides for minimal H1 (mH1) promoter (PAGE purified)
    mH1-Fw: 5’-pAATTCATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGGATTTGGGAATCTTATAAGTTCTGTATGAGAGCACAGA-3’
    mH1-Rv: 5’-pGATCTCTGTGCTCTCATACAGAACTTATAAGATTCCCAAATCCAAAGACATTTCACGTTTATGGTGATTTCCCAGAACACATAGCGACATGCAAATATG-3’
  10. UltraPureTM DNase/RNase-Free distilled water (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10977015 )
  11. Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma-Aldrich, catalog number: M2670-100G )
  12. dNTP set 100 mM solutions (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0181 )
  13. Oligonucleotide primers for mH1 promoter amplification (HPCL purified)
    NbBpu-Fw
    5’-pTTAGGAGTTTTCTCCTAAGCATATTTGCATGTCGCTATGTGTTCTG-3’
    BamHI-Rv
    5’-TGCAGGATCCCTGTGCTCTCATACAGAACTTATAAGATTCCC-3’
  14. Taq DNA polymerase, recombinant (5 U/µl) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EP0402 )
  15. QIAquick PCR Purification Kit (QIAGEN, catalog number: 28106 )
  16. 10x FD buffer (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: B64 )
  17. Nb.Bpu10I (5 U/µl) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: ER1681 )
  18. FastDigest BamHI (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: FD0054 )
  19. shRNA-coding oligonucleotide (PAGE purified) 5’-pGATCTAAAAAGAGCTGTTTCTGAGGAGCCTCTCTTGAAGGCTCCTCAGAAACAGCTCTTTTTA-3’
  20. T4 DNA ligase (5 U/µl) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EL0014 )
  21. Neutralizing oligonucleotide (HPLC purified)
    5’-TTAGGAGTTTTCTCCTAA-3’
  22. Adenosine 5’-triphosphate disodium salt hydrate (Sigma-Aldrich, catalog number: A2383-1G )
  23. FastDigest BglII (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: FD0083 )
  24. T7 DNA polymerase (10 U/µl) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EP0081 )
  25. Phenol solution (Sigma-Aldrich, catalog number: P4557-100ML )
  26. Chloroform (Sigma-Aldrich, catalog number: 288306-1L )
  27. 3-methyl-1-butanol (Sigma-Aldrich, catalog number: 309435-100ML )
  28. Ethanol, absolute (Fisher Scientific, catalog number: BP28184 )
  29. 3 M potassium acetate (pH 4.8)
  30. Sodium acetate (Sigma-Aldrich, catalog number: S2889-250G )
  31. FastDigest EcoRI (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: FD0274 )
  32. Agarose, LE, analytical grade (Promega, catalog number: V3125 )
  33. Ethidium bromide solution (Bio-Rad Laboratories, catalog number: 1610433 )
  34. GeneRuler DNA ladder mix (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: SM0331 )
  35. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888-500G )
  36. Tris-HCl (Powder) (Roche Diagnostics, catalog number: 10812846001 )
  37. EDTA (Sigma-Aldrich, catalog number: EDS-100G )
  38. 10x hybridization buffer (see Recipes)
  39. TE buffer (see Recipes)

Equipment

  1. Pipettes (Gilson, PIPETMAN® Classic, models: P2, P20N, P200N, and P1000N )
  2. Standard thermal cycler (Thermo Fisher Scientific, Applied BiosystemsTM, model: GeneAmp PCR System 9700 )
    Note: This product has been discontinued.
  3. Gel doc (Bio-Rad Gel Doc Imager)
  4. Gel running apparatus (Thermo Fisher Scientific, Amersham BiosciencesTM)
  5. Gel staining tray
  6. Benchtop centrifuge (Eppendorf, model: 5430 R )
  7. Heat block (Eppendorf, model: Thermomixer® Comfort )
  8. Spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDrop 2000 )
  9. Glass beaker (Schott, Duran)
  10. Microwave (Panasonic)

Software

  1. ImageJ (http://imagej.nih.gov/ij/)

Procedure

In this protocol we describe a method for the production of a) shRNA (example db-iPRT-hp-s/as: targeting luciferase) or b) miRNA (example db-hp-miR-125b-1: expressing has-miR-125b-1) expressing minimised dumbbell vectors (Jiang et al., 2016). For these dumbbells, shRNA or miRNA expression is driven by the human minimal H1 (mH1) promoter. The mH1 promoter is PCR-amplified using a forward primer which introduces a cleavage site for a nicking endonuclease and a reverse primer introducing a cleavage site for a conventional restriction endonuclease. After incubating the PCR product with both endonucleases, one loop structure of the dumbbell is formed by the refolding overhang generated by the nicking reaction, whereas the other loop is formed by ligation of a shRNA or miRNA-coding hairpin oligodeoxyribonucleotide. Ligation is performed in the presence of appropriate restriction enzymes to suppress the formation of misligated products (Cost, 2007). The enzymatic ligation assisted by nucleases (ELAN) method suppresses the formation of misligated products such as dimers composed of the loop oligos or the expression cassette which are being cleaved in the presence of restriction endonuclease (in this example BamHI and BglII), thereby facilitating the formation of the intended dumbbell structure which doesn’t comprise the respective endonuclease cleavage sites. Finally, non-ligated DNA is destroyed by exonuclease treatment and exonuclease-resistant dumbbells are purified (Figure 2).


Figure 2. Production strategy for small RNA-expressing minimized dumbbell vectors. The protocol consists of the following steps: First, the mH1 promoter sequence is amplified by PCR. Proper nicking enzyme (NE) and conventional restriction (RE) sites are introduced via the PCR primers. Second, the amplified promoter DNA is digested using the corresponding nicking and restriction enzymes. Third, the digested DNA is purified, and fourth annealed and ligated with the hairpin DNA template oligo using T4 DNA ligase to form an shRNA (left side) or miRNA (right side) expressing dumbbell vector. The addition of a neutralizing oligo and the column purification step significantly improve dumbbell yields. Finally, non-ligated DNA is removed by exonuclease treatment and dumbbell vector DNA is purified using standard DNA purification techniques (Jiang et al., 2016).

  1. Annealing of oligonucleotides
    1. Correct folding of sh/miRNA-coding DNA hairpins is achieved by heating 500 pmol oligo DNA to 95 °C for 3 min (Thermomixer Comfort, Eppendorf) in 10 μl of 1x hybridization buffer and letting the solution cool down to room temperature within 1 h.
    2. The mH1 promoter in this protocol has been modified by introduction of an inverted transcriptional terminator. Therefore, the oligonucleotides mH1-Fw and mH1-Rv are dissolved in nuclease-free distilled water to a concentration of 100 μM. 500 pmol of each strand are annealed in 10 μl 1x hybridization buffer by heating to 95 °C for 3 min (Thermomixer Comfort, Eppendorf) followed by cooling down the solution to room temperature during 1 h (Note 1).

  2. PCR amplification of the mH1 promoter sequence
    1. Add the following components into a thin-walled PCR tube:


    2. Perform PCR using the following thermal cycler conditions:


      PCR product is purified using QIAquick PCR Purification Kit (QIAGEN) following the manufacturer’s recommendations. 

  3. Endonucleolytic cleavage of the PCR product
    1. Combine the reaction components at room temperature in the order indicated:


    2. Incubate the reaction mixture at 37 °C for 4 h, then 85 °C for 5 min to inactivate the enzymes (Note 2). 
    3. Digested DNA is then purified with PCR QIAquick PCR Purification Kit (QIAGEN) following the manufacturer’s recommendations (Note 3).
       
  4. Annealing and ligation of the mH1 promoter and the sh/miRNA-coding hairpin oligonucleotide
    1. Annealing of promoter DNA and sh/miRNA-coding hairpin oligonucleotide is achieved by heating equimolar amounts (80 pmol) of each DNAs in 1x hybridization buffer to 95 °C for 3 min (Thermomixer Comfort, Eppendorf) and letting the solution cool down to room temperature during 1 h.
    2. After annealing, the ligation reaction is setup in the indicated order (Note 4):


    3. Incubate the reaction mixture at 22 °C for 4 h or overnight, then incubate at 80 °C for 10 min to inactivate the enzymes (Note 5).
    4. Withdraw a sample (1-2 µl) from the reaction mixture for gel electrophoresis analysis.

  5. Exonuclease treatment and purification of the dumbbell-shaped vector
    1. Add 1 µl of T7 DNA polymerase (10 U/µl) to the 200 μl ligation mix above, incubate at 37 °C for 1 h and then at 80 °C 10 min to inactivate the enzyme (Note 6).
    2. Withdraw a sample (1-2 µl) from the reaction mixture for gel electrophoresis analysis.
    3. Perform analytical 1.5% agarose gel electrophoresis of the withdrawn samples to monitor the conversion yields and purity of dumbbell vector DNA (Figure 3).
    4. Purify the dumbbell DNA using standard phenol/chloroform extraction followed by ethanol precipitation. In detail, add an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) to the aqueous dumbbell solution, vortex for 30 sec, and separate the aqueous and organic phase by centrifugation (5 min, 13,000 x g). Transfer the upper aqueous phase to a new Eppendorf tube and re-extract residues of phenol. Therefore, add an equal volume of chloroform/isoamyl alcohol (24:1), shake rigorously by hand for 30 sec, and separate the phase by centrifugation (30 sec, 13,000 x g). Transfer the upper aqueous phase to a new Eppendorf tube and repeat the re-extraction process twice (Note 7). For ethanol precipitation, the aqueous phase was topped up to 400 µl with distilled water, then 0.1 volumes (40 µl) of 3 M potassium acetate (pH 4.8) followed by 2.5 volumes (1,100 µl) ethanol were added, the solution was mixed and incubated at -20 °C for 20 min. DNA was pelleted by centrifugation at 13,000 x g, 4 °C for 15 min. The pellets were then air-dried. Dissolve the purified DNA in TE buffer or distilled water.


      Figure 3. Analytical gel electrophoresis of dumbbell vector DNA after different treatments. Additional treatments increased dumbbell conversion yield: basic protocol (1), basic protocol with neutralizing oligo (50 pmol) (2), basic protocol with column purification (using the QIAgen PCR Purification Kit and following standard protocol by QIAgen) (3), and basic protocol with both treatments (4). The neutralizing oligo binds to the oligo released after nicking enzyme cleavage and prevents that this can bind again to the generated overhang. The column purification removes the small cleavage products and prevents them from relegation in the subsequent ligation step. The highest conversion yield (91%) was achieved with the additional of column purification step. The conversion yield is defined as the yield obtained when comparing the size of the expected dumbbell before and after exonuclease treatment. CP, column purification; Neu, neutralizing oligo (Jiang et al., 2016).

Data analysis

Analysis of dumbbell DNA conversion yield:

  1. Measure the intensity of the DNA bands corresponding to the dumbbell products in the electrophoresis gel with ImageJ using the wand tool.
  2. Conversion yield is calculated by dividing the intensity of the band after exonuclease treatment by that of the corresponding band in the ligation sample.

Notes

  1. Alternatively, the mH1 sequence can be generated through PCR amplification from plasmid DNA pSuper-mH1 which was constructed by cloning the hybridized oligonucleotides into the pSuper-basic plasmid. To insert the annealed sequence into the pSuper-basic plasmid, the vector (1 µg) was first digested with EcoRI and BglII at 37 °C for 4 h. After digestion, the DNA was purified using the QIAgen PCR Purification Kit. Ligation was performed using equimolar amounts (0.25 pmol) of the digested vector backbone and annealed insert at 22 °C for 4 h. The correct clone was confirmed by sequencing. If pSuper-mH1 was used for the PCR reaction, 10 ng was used as the template.
  2. We used Nb.Bpu10I as the nicking enzyme based on a previous report by Taki et al. (2004). The enzyme is supplied with 10x buffer R but here the universal 10x buffer FD was used instead after consulting Thermo Fisher’s technical support.
  3. Although the digestion product from this step can directly be used for ligation, we found an additional purification step could greatly increase the dumbbell conversion yield as shown in Figure 2.
  4. In some cases, a neutralising oligo was added into the ligation reaction to suppress reannealing of the short nicking fragment (see Figure 2). Ligation was performed in FD buffer complemented with 1 mM final ATP instead of the ligation buffer since all enzymes including the ligase were 100% active in this buffer. To ligate the sh/miRNA-coding hairpin template and the promoter DNA, we followed the enzymatic ligation assisted by nucleases (ELAN) technique as described before (Cost, 2007). The sh/miRNA-coding hairpin oligonucleotides are ordered PAGE purified and can directly be used for ligation.
  5. According to our experience, the ligation reaction was completed within 4 h and longer incubation times did not improve the dumbbell yield.
  6. T7 DNA polymerase exhibits 100% activity in the FD buffer and was therefore directly added into the ligation mixture.
  7. It is essential to shake by hand instead of vortexing in order to mix the phase efficiently.

Recipes

  1. 10x hybridization buffer
    1 M NaCl
    100 mM MgCl2
    200 mM Tris-HCl, pH 7.4
  2. TE buffer
    10 mM Tris-HCl, pH 8.0
    1 mM EDTA

Acknowledgments

The protocol described herein was developed and utilized previously in Jiang et al. (2016). This work was supported by the National University of Singapore [Bridging Grant NUHSRO/2015/091/Bridging/02], the National Medical Research Council of Singapore [New Investigator Grant number NMRC/NIG/1058/2011], and the Ministry of Education of Singapore [Academic Research Fund (AcRF) Tier 1 Faculty Research Committee (FRC) grants number T1-2011Sep-04 and T1-2014Apr-02 and Seed Fund for Basic Science Research number T1-BSRG 2015-05], all to VP. The authors declare competing financial interests. A patent application covering major parts of the work is pending.

References

  1. Chen, Z. Y., He, C. Y., Meuse, L. and Kay, M. A. (2004). Silencing of episomal transgene expression by plasmid bacterial DNA elements in vivo. Gene Ther 11(10): 856-864.
  2. Chen, Z. Y., Riu, E., He, C. Y., Xu, H. and Kay, M. A. (2008). Silencing of episomal transgene expression in liver by plasmid bacterial backbone DNA is independent of CpG methylation. Mol Ther 16(3): 548-556.
  3. Cost, G. J. (2007). Enzymatic ligation assisted by nucleases: simultaneous ligation and digestion promote the ordered assembly of DNA. Nat Protoc 2(9): 2198-2202.
  4. Jiang, X., Yu, H., Teo, C. R., Tan, G. S., Goh, S. C., Patel, P., Chua, Y. K., Hameed, N. B., Bertoletti, A. and Patzel, V. (2016). Advanced design of dumbbell-shaped genetic minimal vectors improves non-coding and coding RNA expression. Mol Ther 24(9): 1581-1591.
  5. Nicol, F., Wong, M., MacLaughlin, F. C., Perrard, J., Wilson, E., Nordstrom, J. L. and Smith, L. C. (2002). Poly-L-glutamate, an anionic polymer, enhances transgene expression for plasmids delivered by intramuscular injection with in vivo electroporation. Gene Ther 9(20): 1351-1358.
  6. Schakowski, F., Gorschluter, M., Buttgereit, P., Marten, A., Lilienfeld-Toal, M. V., Junghans, C., Schroff, M., Konig-Merediz, S. A., Ziske, C., Strehl, J., Sauerbruch, T., Wittig, B. and Schmidt-Wolf, I. G. (2007). Minimal size MIDGE vectors improve transgene expression in vivo. In Vivo 21(1): 17-23.
  7. Schakowski, F., Gorschluter, M., Junghans, C., Schroff, M., Buttgereit, P., Ziske, C., Schottker, B., Konig-Merediz, S. A., Sauerbruch, T., Wittig, B. and Schmidt-Wolf, I. G. (2001). A novel minimal-size vector (MIDGE) improves transgene expression in colon carcinoma cells and avoids transfection of undesired DNA. Mol Ther 3(5 Pt 1): 793-800.
  8. Taki, M., Kato, Y., Miyagishi, M., Takagi, Y. and Taira, K. (2004). Small-interfering-RNA expression in cells based on an efficiently constructed dumbbell-shaped DNA. Angew Chem Int Ed Engl 43(24): 3160-3163

简介

使用非病毒载体进行基因治疗的主要障碍是在postmitotic组织中转基因表达的持续时间短。以前的研究表明,即使载体DNA被保留,传代质粒的转基因表达也在递送后不久就下降到亚治疗水平,提示转录在体内沉默(Nicol等人)。 ,2002; Chen等人,2004)。新出现的证据表明质粒细菌骨架序列负责转录抑制,该过程与CpG甲基化无关(Chen等人,2008)。仅开发了用于转基因表达的必需元件的哑铃型DNA载体已被开发出来,以规避这些缺点。已经显示这种新的非病毒载体在体外和体内改善转基因表达(Schakowski等人,2001和2007)。在这里我们描述一种快速有效地生产最小化的表达小RNA的哑铃载体的新方法。简言之,将PCR扩增的启动子序列连接到化学合成的发夹RNA编码DNA模板以形成共价闭合的哑铃载体。这种新技术可以促进哑铃型载体用于临床前研究和人类基因治疗的应用。

背景 关于递送,小的载体大小有利地改善细胞外转运,包括通过细胞外基质网络的外渗和扩散以及细胞摄取和核扩散。已经开发了各种哑铃载体生产方法,包括产生表达小RNA的哑铃的方法,例如小发夹RNA(shRNA)和微小RNA(miRNA)(Schakowski等人,2001; Taki等人,2004)。这些载体通常含有冗余的序列,因为表达的RNA是自身互补的。我们消除了产生最小化哑铃向量的冗余序列,其中转录围绕哑铃本身的发夹结构(Jiang等人,2016)。这种最小化的哑铃载体可以短至130bp,代表所报告的最小表达载体。常规质粒,含有线性表达盒的哑铃和新型发夹模板转录哑铃载体之间的说明性比较显示在图1中。该新方案有助于生成新的最小化的小RNA表达哑铃载体。


图1.小发夹RNA表达质粒和哑铃载体的结构上两个:常规质粒p-iPR-linear-s / as和哑铃db-iPR-linear-s /作为具有线性的载体shRNA表达盒和整合的启动子限制性内切核酸酶位点元件(iPR)。较低的矢量:最小化发夹模板(hp)哑铃拥有iPRT元素。 R表示限制性突出连接位点。 T表示终止信号。 IT指示反向终止信号。环路L1和L2是(T)四环四环。

关键字:哑铃形载体, 最小载体, 小分子RNA表达, miRNA, shRNA, 基因疗法

材料和试剂

  1. 0.2-10μl移液器吸头,Corning ® Isotip ®过滤(Corning,目录号:4807)
  2. 1-200μl移液器吸头(Corning,Axyge ®,目录号:TF-200-R-S)
  3. 100-1,000μl移液器吸头(Corning,Axygen ®,目录号:TF-1000-R-S)
  4. 1.5ml微量离心管(RNase,DNase和无热原)(Corning,Axygen,目录号:MCT-150-C)
  5. 0.2ml薄壁PCR管(Thermo Fisher Scientific,Thermo Scientific TM,目录号:3412)
  6. Falcon ®将50ml锥形离心管(Corning,Falcon ®,目录号:352070)
  7. pSuper基本载体(Oligoengine,目录号:VEC-pBS-0002)
  8. miRNA编码寡核苷酸(PAGE纯化)5'-pGATCTAGCACGACTCGCAGCTCCCAAGAGCCTAACCCGTGGATTTAAACGGTAAACATCACAAGTTAGGGTCTCAGGGACTGAGAGGAGCGCAA-3'
  9. 用于最小H1(mH1)启动子的寡核苷酸(PAGE纯化)
    mH1-Fw:5'-pAATTCATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGGATTTGGGAATCTTATAAGTTCTGTATGAGAGCACAGA-3'
    mH1-Rv:5'-pGATCTCTGTGCTCTCATACAGAACTTATAAGATTCCCAAATCCAAAGACATTTCACGTTTATGGTGATTTCCCAGAACACATAGCGACATGCAAATATG-3'
  10. UltraPure TM DNase / RNase-Free蒸馏水(Thermo Fisher Scientific,Invitrogen TM,目录号:10977015)
  11. 氯化镁六水合物(MgCl 2·6H 2 O)(Sigma-Aldrich,目录号:M2670-100G)
  12. dNTP设置100mM溶液(Thermo Fisher Scientific,Thermo Scientific TM,目录号:R0181)
  13. 用于mH1启动子扩增的寡核苷酸引物(HPCL纯化)
    NbBpu-Fw
    5'-pTTAGGAGTTTTCTCCTAAGCATATTTGCATGTCGCTATGTGTTCTG-3'
    BamHI-Rv
    5'-TGCAGGATCCCTGTGCTCTCATACAGAACTTATAAGATTCCC-3'
  14. DNA聚合酶,重组体(5U /μl)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:EP0402)
  15. QIAquick PCR纯化试剂盒(QIAGEN,目录号:28106)
  16. 10x FD缓冲液(Thermo Fisher Scientific,Thermo Scientific TM,目录号:B64)
  17. Nb.Bpu10I(5U /μl)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:ER1681)
  18. FastDigest Bam(Thermo Fisher Scientific,Thermo Scientific TM,目录号:FD0054)
  19. shRNA编码寡核苷酸(PAGE纯化)5'-pGATCTAAAAAGAGCTGTTTCTGAGGAGCCTCTCTTGAAGGCTCCTCAGAAACAGCTCTTTTTA-3'
  20. T4 DNA连接酶(5U /μl)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:EL0014)
  21. 中和寡核苷酸(HPLC纯化)
    5'-TTAGGAGTTTTCTCCTAA-3'
  22. 腺苷5'-三磷酸二钠盐水合物(Sigma-Aldrich,目录号:A2383-1G)
  23. FastDigest II(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:FD0083)
  24. T7 DNA聚合酶(10U /μl)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:EP0081)
  25. 苯酚溶液(Sigma-Aldrich,目录号:P4557-100ML)
  26. 氯仿(Sigma-Aldrich,目录号:288306-1L)
  27. 3-甲基-1-丁醇(Sigma-Aldrich,目录号:309435-100ML)
  28. 乙醇,绝对(Fisher Scientific,目录号:BP28184)
  29. 3M醋酸钾(pH 4.8)
  30. 乙酸钠(Sigma-Aldrich,目录号:S2889-250G)
  31. FastDigest RI(Thermo Fisher Scientific,Thermo Scientific TM,目录号:FD0274)
  32. 琼脂糖,LE,分析纯(Promega,目录号:V3125)
  33. 溴化乙锭溶液(Bio-Rad Laboratories,目录号:1610433)
  34. GeneRuler DNA梯形混合物(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:SM0331)
  35. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S9888-500G)
  36. Tris-HCl(Powder)(Roche Diagnostics,目录号:10812846001)
  37. EDTA(Sigma-Aldrich,目录号:EDS-100G)
  38. 10x杂交缓冲液(参见食谱)
  39. TE缓冲(见配方)

设备

  1. 移液器(Gilson,PIPETMAN ®经典型号:P2,P20N,P200N和P1000N)
  2. 标准热循环仪(Thermo Fisher Scientific,Applied Biosystems TM,型号:GeneAmp PCR System 9700)
    注意:本产品已停产。
  3. Gel doc(Bio-Rad Gel Doc Imager)
  4. 凝胶运行装置(Thermo Fisher Scientific,Amersham Biosciences TM
  5. 凝胶染色托盘
  6. 台式离心机(Eppendorf,型号:5430 R)
  7. 热块(Eppendorf,型号:Thermomixer ®舒适)
  8. 分光光度计(Thermo Fisher Scientific,Thermo Scientific TM,型号:NanoDrop 2000)
  9. 玻璃烧杯(Schott,Duran)
  10. 微波(松下)

软件

  1. ImageJ( http://imagej.nih.gov/ij/

程序

在本协议中,我们描述了a)shRNA(例如db-iPRT-hp-s / as:靶向荧光素酶)或b)miRNA(例如db-hp-miR-125b-1:表达has-miR- 125b-1)表达最小化的哑铃载体(Jiang等人,2016)。对于这些哑铃,shRNA或miRNA表达由人类最小H1(mH1)启动子驱动。使用引入用于切口内切核酸酶的切割位点的正向引物和引入常规限制性内切核酸酶切割位点的反向引物对mH1启动子进行PCR扩增。在PCR产物与内切核酸酶两者孵育后,通过切口反应产生的重折叠突出形成哑铃的一个环结构,而另一个环通过连接shRNA或miRNA编码的发夹寡脱氧核糖核苷酸形成。在适当的限制酶存在下进行连接,以抑制产生不必要的产物(Cost,2007)。通过核酸酶(ELAN)方法辅助的酶促连接抑制了在限制性内切核酸酶存在下被切割的环寡聚体或表达盒组成的失调产物如二聚体的形成(在该实施例中, HI和Bgl II),从而有助于形成不包含相应的内切核酸酶切割位点的预期哑铃结构。最后,通过外切核酸酶处理破坏未连接的DNA,并纯化外切核酸酶哑铃(图2)。


图2.表达小RNA的最小化哑铃载体的生产策略。该方案包括以下步骤:首先,通过PCR扩增mH1启动子序列。通过PCR引物引入适当的切口酶(NE)和常规限制(RE)位点。第二,使用相应的切口和限制酶消化扩增的启动子DNA。第三,将经消化的DNA纯化,并用T4DNA连接酶进行第四次退火并与发夹DNA模板寡核苷酸连接,形成shRNA(左侧)或miRNA(右侧)表达哑铃载体。添加中和寡核苷酸和柱纯化步骤显着提高了哑铃产量。最后,通过外切核酸酶处理除去未连接的DNA,并使用标准DNA纯化技术纯化哑铃载体DNA(Jiang等人,2016)。

  1. 寡核苷酸退火
    1. 通过在10μl1x杂交缓冲液中加热500pmol寡核苷酸至95℃3分钟(Thermomixer Comfort,Eppendorf)并使溶液在1小时内冷却至室温来实现sh / miRNA编码DNA发夹的正确折叠。
    2. 该方案中的mH1启动子已经通过引入反向转录终止子而被修饰。因此,将寡核苷酸mH1-Fw和mH1-Rv溶于无核酸酶的蒸馏水中至浓度为100μM。通过加热至95℃3分钟(Thermomixer Comfort,Eppendorf),在10μl1x杂交缓冲液中退火500pmol,然后在1小时内将溶液冷却至室温(注1)。

  2. mH1启动子序列的PCR扩增
    1. 将以下组件添加到薄壁PCR管中:


    2. 使用以下热循环仪条件进行PCR:


      按照制造商的建议,使用QIAquick PCR Purification Kit(QIAGEN)纯化PCR产物。 

  3. PCR产物的内切核酸切割
    1. 在室温下按照所示顺序合并反应组分:


    2. 将反应混合物在37℃孵育4小时,然后在85℃孵育5分钟以灭活酶(注2)。 
    3. 然后根据制造商的建议(注3),用PCR QIAquick PCR Purification Kit(QIAGEN)纯化消化的DNA。
       
  4. mH1启动子和sh / miRNA编码的发夹寡核苷酸的退火和连接
    1. 启动子DNA和sh / miRNA编码发夹寡核苷酸的退火是通过将1x杂交缓冲液中的每种DNA等摩尔量(80pmol)加热至95℃3分钟(Thermomixer Comfort,Eppendorf)并使溶液冷却至室1小时内温度。
    2. 退火后,连接反应按照指示顺序设置(注4):


    3. 将反应混合物在22℃孵育4小时或过夜,然后在80℃下孵育10分钟以灭活酶(注5)。
    4. 从反应混合物中取出样品(1-2μl)进行凝胶电泳分析
  5. 外来核酸酶处理和纯化哑铃形载体
    1. 在上述200μl连接混合物中加入1μlT7 DNA聚合酶(10 U /μl),37℃孵育1 h,然后在80°C孵育10 min,使酶失活(注6)。
    2. 从反应混合物中取出样品(1-2μl)进行凝胶电泳分析
    3. 对取出的样品进行分析性1.5%琼脂糖凝胶电泳,以监测哑铃载体DNA的转化产率和纯度(图3)。
    4. 使用标准苯酚/氯仿提取纯化哑铃DNA,然后乙醇沉淀。详细地,将等体积的苯酚/氯仿/异戊醇(25:24:1)加入到水性哑铃溶液中,旋涡30秒,并通过离心(5分钟,13,000xg)分离水相和有机相)。将上层水相转移到新的Eppendorf管中,并重新提取苯酚残留物。因此,加入等体积的氯仿/异戊醇(24:1),用手严格摇动30秒,并通过离心(30秒,13,000×g / g)分离相。将上层水相转移到新的Eppendorf管中,并重复两次重萃取过程(注7)。对于乙醇沉淀,用蒸馏水将水相加至400μl,然后加入0.1体积(40μl)3M乙酸钾(pH 4.8),然后加入2.5体积(1,100μl)乙醇,将溶液混合,在-20℃下孵育20分钟。通过以13,000 x g,4℃离心15分钟使DNA沉淀。然后将颗粒风干。将纯化的DNA溶解在TE缓冲液或蒸馏水中

      图3.不同处理后哑铃载体DNA的分析凝胶电泳。其他处理增加了哑铃转换产率:基本方案(1),中和寡聚(50 pmol)的基本方案(2),基本方案与柱纯化(使用QIAgen PCR纯化试剂盒和QIAgen的以下标准方案)(3)和两种处理的基本方案(4)。中和寡聚体结合在切割酶切割后释放的寡核苷酸,并防止这可能再次结合到产生的突出端。柱纯化除去小切割产物并防止它们在随后的连接步骤中降级。通过另外的柱纯化步骤实现了最高的转化率(91%)。转化产率定义为当比较核酸外切酶处理前后预期哑铃的大小时获得的产量。 CP,柱纯化; Neu,中和寡核苷酸(Jiang等人,2016)。

数据分析

哑铃DNA转化产率分析:

  1. 使用魔杖工具使用ImageJ测量电泳凝胶中与哑铃产品相对应的DNA条带的强度。
  2. 通过将外切核酸酶处理后的条带的强度除以连接样品中相应条带的强度来计算转化产率。

笔记

  1. 或者,可以通过从质粒DNA pSuper-mH1的PCR扩增产生mH1序列,其通过将杂交的寡核苷酸克隆到pSuper碱性质粒中构建。为了将退火的序列插入pSuper碱性质粒,首先用EcoRI和BglⅡ在37℃下消化载体(1μg)4小时。消化后,使用QIAgen PCR Purification Kit纯化DNA。使用等摩尔量(0.25pmol)消化的载体骨架进行连接,并在22℃退火插入物4小时。通过测序确认正确的克隆。如果使用pSuper-mH1进行PCR反应,则使用10ng作为模板
  2. 根据Taki等人先前的报告,我们使用Nb.Bpu10I作为切口酶。 (2004年)。酶供应10倍缓冲液R,但是在咨询了赛默飞世尔技术支持后,使用通用的10倍缓冲液FD。
  3. 虽然这一步骤的消化产物可以直接用于连接,我们发现一个额外的纯化步骤可以大大提高哑铃转化率,如图2所示。
  4. 在一些情况下,将中和寡核苷酸加入到连接反应中以抑制短切片段的再退火(参见图2)。在用1mM最终ATP补充的FD缓冲液中进行连接,而不是连接缓冲液,因为包括连接酶的所有酶在该缓冲液中为100%活性。为了连接sh / miRNA编码的发夹模板和启动子DNA,我们按照如前所述的核酸酶(ELAN)技术进行酶连接辅助(Cost,2007)。将sh / miRNA编码的发夹寡核苷酸序列PAGE纯化,可直接用于连接
  5. 根据我们的经验,连接反应在4小时内完成,较长的孵育时间没有提高哑铃产量。
  6. T7 DNA聚合酶在FD缓冲液中显示出100%的活性,因此直接加入到连接混合物中
  7. 必须用手摇动,而不是旋涡,以便有效地混合相位。

食谱

  1. 10x杂交缓冲液
    1 M NaCl
    100mM MgCl 2
    200mM Tris-HCl,pH7.4
  2. TE缓冲区
    10mM Tris-HCl,pH8.0
    1 mM EDTA

致谢

这里描述的协议在以前在Jiang等人开发和使用。 (2016)。新加坡国立大学新加坡国立医科大学附属新加坡国立大学新加坡国立医学研究委员会(新加坡国立医学研究理事会,新加坡国立医科大学核医学研究中心,新加坡国立医科大学,尼日利亚国立大学,尼日利亚共和国),以及教育部新加坡[学术研究基金(AcRF)第1级教师研究委员会(FRC)授予T1-2011Sep-04和T1-2014Apr-02以及“基础科学研究种子基金”号T1-BSRG 2015-05]。作者宣称相互竞争的经济利益。涵盖主要工作的专利申请正在等待。

参考

  1. Chen,ZY,He,CY,Meuse,L.和Kay,MA(2004)。通过质粒细菌DNA元件在体内沉默附加型转基因 Gene Ther 11(10):856-864。
  2. Chen,ZY,Riu,E.,He,CY,Xu,H. and Kay,MA(2008)。通过质粒细菌骨架DNA沉默肝脏中的附加型转基因表达与CpG甲基化无关。Mol Ther 16(3):548 -556。
  3. 成本,GJ(2007)。由核酸酶辅助的酶结合:同时连接和消化促进DNA的有序组装。 Nat Protoc 2(9):2198-2202。
  4. 江泽民,俞,H.,曹,谭,谭,GS,Goh,SC,Patel,P.,Chua,YK,Hameed,NB,Bertoletti,A。和Patzel,V。(2016) 哑铃型遗传最小载体的高级设计改善了非编码和编码RNA表达。 Mol Ther 24(9):1581-1591。
  5. Nicol,F.,Wong,M.,MacLaughlin,FC,Perrard,J.,Wilson,E.,Nordstrom,JL和Smith,LC(2002)。< a class =“ke-insertfile”href =“http ://www.ncbi.nlm.nih.gov/pubmed/12365000“target =”_ blank“>阴离子聚合物聚-L-谷氨酸增强体内肌内注射传递的质粒的转基因表达, em> electroporation。 Gene Ther 9(20):1351-1358。
  6. Schakowski,F.,Gorschluter,M.,Buttgereit,P.,Marten,A.,Lilienfeld-Toal,MV,Junghans,C.,Schroff,M.,Konig-Merediz,SA,Ziske,C.,Strehl,J 。,Sauerbruch,T.,Wittig,B.和Schmidt-Wolf,IG(2007)。< a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed / 17354609“target =”_ blank“>最小尺寸MIDGE载体在体内改善转基因表达。在体内 21(1):17-23。
  7. Schakowski,F.,Gorschluter,M.,Junghans,C.,Schroff,M.,Buttgereit,P.,Ziske,C.,Schottker,B.,Konig-Merediz,SA,Sauerbruch,T.,Wittig,和Schmidt-Wolf,IG(2001)。一本小说最小尺寸载体(MIDGE)改善结肠癌细胞中的转基因表达,并避免转染不需要的DNA。 Mol Ther 3(5 Pt 1):793-800。
  8. Taki,M.,Kato,Y.,Miyagishi,M.,Takagi,Y.和Taira,K。(2004)。基于有效构建的哑铃形DNA的细胞中的小干扰RNA表达。 Angew Chem Int Ed Engl 43(24):3160-3163
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Jiang, X. and Patzel, V. (2017). Formation of Minimised Hairpin Template-transcribing Dumbbell Vectors for Small RNA Expression. Bio-protocol 7(11): e2313. DOI: 10.21769/BioProtoc.2313.
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