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Advanced Design of Minimalistic Dumbbell-shaped Gene Expression Vectors
极简化哑铃形基因表达载体的优化设计   

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Abstract

Minimal DNA vectors exclusively comprising therapeutically relevant sequences hold great promise for the development of novel therapeutic regimen. Dumbbell-shaped vectors represent non-viral non-integrating DNA minimal vectors which have entered an advanced stage of clinical development (Hardee et al., 2017). Spliceable introns and DNA nuclear import signals such as SV40 enhancer sequences are molecular features that have found multiple applications in plasmid vectors to improve transgene expression. In dumbbells however, effects triggered by introns were not investigated and DNA-based nuclear import sequences have not found applications yet, presumably because dumbbell vectors have continuously been minimized with regard to size. We investigated the effects of an intron and/or SV40 enhancer derived sequences on dumbbell vector driven reporter gene expression. The implementation of a spliceable intron was found to enhance gene expression unconditionally in all investigated cell lines. Conversely, the use of the SV40 enhancer improved gene expression in a cell type-dependent manner. Though both features significantly enlarge dumbbell vector size, neither the intron nor the enhancer or a combination of both revealed a negative effect on gene expression. On the contrary, both features together improved dumbbell-driven gene expression up to 160- or 56-fold compared with plasmids or control dumbbells. Thus, it is highly recommended to consider an intron and the SV40 enhancer for dumbbell vector design. Such an advanced design can facilitate pre-clinical and clinical applications of dumbbell-shaped DNA vectors.

Keywords: Dumbbell vector(哑铃形载体), Minimal DNA vector(最小DNA载体), Transgene expression(转基因表达), Genetic therapy(基因疗法), Intron(内含子), SV40 enhancer(SV40增强子)

Background

Although many genes have been expressed using dumbbell-shaped DNA vectors, most of these applications used the basic design comprising a promoter, the coding sequence (CDS), and a transcriptional terminator. Some vectors included a chimeric intron, however, it was not reported whether transgene expression was enhanced by this design (Schirmbeck et al., 2001). Here we studied the effects triggered by molecular features that frequently find applications in plasmid design on dumbbell-driven gene expression: 1. A chimeric intron derived from the human β-globin gene–splicing is known to facilitate RNA processing, nuclear export, and subsequently gene expression (Luo and Reed, 1999); and 2. The Simian virus 40 (SV40) enhancer which can enhance the activity of the homologous SV40 promoter and which in addition was reported to function as an active DNA nuclear import sequence (Dean, 1997; Dean et al., 1999). The proposed mechanism behind this phenomenon is that the SV40 enhancer recruits transcription factors harboring protein nuclear import signals in the cytoplasm and that the vector DNA is piggyback translocated into the nucleus with support of the protein nuclear import machinery. We generated luciferase-expressing dumbbell vectors harboring either both, only one or none of these molecular features and monitored transgene expression in HEK293T and HepG2 cells. Our data demonstrate that introns and the SV40 enhancer can substantially improve dumbbell vector design (Jiang et al., 2016).

Materials and Reagents

  1. Pipette tips (Corning® Isotip® filtered 0.2-10 μl) (Corning, catalog number: 4807 )
  2. Pipette tips (Axygen® TF200RS 1-200 μl) (Corning, Axygen®, catalog number: TF-200-R-S )
  3. Pipette tips (Axygen® TF1000RS 100-1,000 μl) (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. FalconTM 50 ml conical centrifuge tubes (Corning, Falcon®, catalog number: 352070 )
  7. T-75 flask (Corning, catalog number: 3290 )
  8. 24-well cell culture plate (Corning, catalog number: 3527 )
  9. 96-well plate (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 442404 )
  10. HEK293T cells (ATCC, catalog number: CRL-3216 )
  11. HepG2 cells (ATCC, catalog number: HB-8065 )
  12. pGL3-control vector (Promega, catalog number: E1741 )
  13. One Shot® TOP10 Chemically Competent E. coli (Thermo Fisher Scientific, InvitrogenTM, catalog number: C404010 )
  14. Chimeric human β-globin intron sequence (Gene synthesis, GeneArt, Applied Biosystems): 5’-CAGGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACAGAGACGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGG-3’
  15. SV40 enhancer sequence: 5’-CGATGGAGCGGAGAATGGGCGGAACTGGGCGGAGTTAGGGGCGGGATGGGCGGAGTTAGGGGCGGGACTATGGTTGCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGCTGGGGAGCCTGGGGACTTTCCACACCTGGTTGCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGCTGGGGAGCCTGGGGACTTTCCACACCCTAACTGACACACATTCCACAGC-3’
  16. Oligonucleotide primers for chimeric intron amplification (Synthesized by Integrated DNA Technologies, 25 nmol scale, deprotected desalted):
    intron-Fw: 5’-ATCTATCGGGATCCAAGCTTCAGGTAAGTATCAAGGTTACAAGACAGG-3’
    intron-Rv: 5’-CATTATCTGGATCCCCATGGACCCTGTGGAGAGAAAGGCAA-3’
  17. Loop sequences (Synthesized by Integrated DNA Technologies, 25 nmol scale, deprotected desalted):
    Loop-1: 5’-pGATCTGACCAGTTTTCTGGTCA-3’
    Loop-2: 5’-pTCGACAGGCTCTTTTGAGCCTG-3’
  18. Oligonucleotide primers for poly(A) signal (Synthesized by Integrated DNA Technologies, 25 nmol scale, deprotected desalted):
    polyA-Fw: 5’-TGTAATTCTAGAGTCGGGGCG-3’
    polyA-Rv: 5’-ATCTATCGGGATCCTTACCACATTTGTAGAGGTT-3’
  19. FastDigest HindIII (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: FD0504 )
  20. FastDigest NcoI (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: FD0573 )
  21. FastDigest BamHI (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: FD0054 )
  22. FastDigest BglII (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: FD0083 )
  23. FastDigest XhoI (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: FD0694 )
  24. FastDigest SalI (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: FD0644 )
  25. FastDigest AseI (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: FD0914 )
  26. UltraPureTM DNase/RNase-free distilled water (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10977015 )
  27. T7 DNA polymerase (10 U/µl) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EP0081 )
  28. Taq DNA polymerase, recombinant (5 U/µl) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EP0402 )
  29. 10x FD buffer (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: B64 )
  30. T4 DNA ligase (5 U/µl) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EL0014 )
  31. dNTP set 100 mM solutions (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0181 )
  32. QIAquick PCR purification kit (QIAGEN, catalog number: 28106 )
  33. Potassium acetate (Sigma-Aldrich, catalog number: P1190-100G )
  34. Agarose, LE, analytical grade (Promega, catalog number: V3125 )
  35. Ethanol, absolute (Fisher Scientific, catalog number: BP28184 )
  36. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888-500G )
  37. Magnesium chloride hexachloride (MgCl2·6H2O) (Sigma-Aldrich, catalog number: M2670-100G )
  38. Tris-HCl (Roche Diagnostics, catalog number: 10812846001 )
  39. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: EDS-100G )
  40. Adenosine 5’-triphosphate disodium salt hydrate (Sigma-Aldrich, catalog number: A2383-1G )
  41. Ethidium bromide solution (Bio-Rad Laboratories, catalog number: 1610433 )
  42. Phenol solution (Sigma-Aldrich, catalog number: P4557-100ML )
  43. Chloroform (Sigma-Aldrich, catalog number: 288306-1L )
  44. 3-Methyl-1-butanol (Sigma-Aldrich, catalog number: 309435-100ML )
  45. GeneRuler DNA Ladder Mix (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: SM0331 )
  46. HyClone Dulbecco’s modified Eagles medium/high glucose with L-glutamine, sodium pyruvate (GE Healthcare, HyCloneTM, catalog number: SH30243.01 )
  47. HyClone Standard Fetal Bovine Serum (GE Healthcare, HyCloneTM, catalog number: SH30088.03 )
  48. Penicillin-streptomycin (10,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  49. Lipofectamine® 2000 Transfection Reagent (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11668019 )
  50. Opti-MEM® I Reduced Serum Medium (Thermo Fisher Scientific, GibcoTM, catalog number: 31985070 )
  51. Luciferase Assay System (Promega, catalog number: E1501 )
  52. Liquid nitrogen
  53. TE buffer (see Recipes)

Equipment

  1. Pipettes (Gilson, PIPETMAN Classic®, P2 , P20N , P200N , and P1000N )
  2. CO2 incubator (Thermo Electron)
  3. Shaker (Heidolph Instruments, model: Unimax 2010 )
  4. Glass beaker (Schott, Duran)
  5. Standard thermal cycler (Thermo Fisher Scientific, Applied BiosystemsTM, model: GeneAmp PCR System 9700 )
    Note: This product has been discontinued.
  6. Gel doc (Bio-Rad Laboratories, Gel Doc Imager)
  7. Gel running apparatus (Amersham Biosciences)
  8. Gel staining tray (GE Healthcare)
  9. Benchtop centrifuge (Eppendorf, model: 5430 R )
  10. Heat block (Thermomixer comfort) (Eppendorf)
  11. Spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 2000 )
  12. Microwave (Panasonic)
  13. Class II Biological Safety Cabinet (Gelman)
  14. Synergy H1 Multi-Mode Reader (BioTek Instruments, model: Synergy H1 )

Software

  1. GraphPad Prism software 5.0

Procedure

  1. Design and molecular cloning of luciferase-expressing plasmid variants
    To study the function of a synthetic intron and/or the SV40 enhancer on dumbbell-driven gene expression, we clone the corresponding plasmid vectors based on the pGL3-control vector. The pGL3-control vector harbors the firefly luciferase gene under the control of the SV40 promoter together with the SV40 enhancer element at the 3’ end of the expression cassette. Cloning is done as described below:
    1. The 137 bp chimeric human β-globin mini-intron is synthesized by gene synthesis (GeneArt, Applied Biosystems), PCR amplified using primers intron-Fw and intron-Rv, and then inserted into the pGL3-control plasmid using the HindIII and NcoI sites to create a luciferase expression vector featured with both, an intron and the SV40 enhancer (int-luc-enh) (Jiang et al., 2016 and 2017). An illustration of the production process is shown in Figure 1.


      Figure 1. Illustration of the plasmid cloning schemes. Step 1: Plasmid p-int-luc-enh is derived by insertion of the b-globin mini-intron into the HindIII and NcoI sites of vector pGL3-Control (p-luc-enh). Step 2: Plasmid p-luc can be generated by exchanging the SV40 enhancer together with the SV40 poly(A) site of vector pGL3-Control (p-luc-enh) by the SV40 polyA site using XbaI and BamHI. Step 3: Plasmid p-int-luc is cloned by exchanging the SV40 enhancer together with the SV40 polyA site of vector p-int-luc-enh by the SV40 polyA site using XbaI and BamHI.

    2. The SV40 enhancer sequence in pGL3-control is deleted by digestion of the vector with XbaI and BamHI. By doing so, we also delete the SV40 late poly(A) signal. To retrieve the SV40 poly(A) signal, its sequence is PCR-amplified using the primers polyA-Fw and polyA-Rv which introduce the XbaI and BamHI sites. The PCR product is cleaved with XbaI and BamHI and the SV40 poly (A) signal is re-inserted to create a plasmid lacking both, the intron and the SV40 enhancer (luc) (Jiang et al., 2016 and 2017). An illustration of the production process is shown in Figure 1.
    3. The equivalent procedure described under step A2 is repeated starting with the pGL3-control variant harboring the intron and the SV40 enhancer to generate a plasmid that is only featured with the intron (int-luc) (Jiang et al., 2016). An illustration of the production process is shown in Figure 1.
    4. For simplification, novel names are assigned to the constructs as listed in Table 1.

      Table 1. Nomenclature of luciferase expression constructs. Plasmids were assigned the prefix ‘p’, dumbbells the prefix ‘db’.


  2. Production of luciferase-expressing dumbbell vectors
    1. Luciferase-expressing dumbbell vectors are produced from the corresponding plasmids described under Procedure A. using the enzymatic ligation assisted by nucleases (ELAN) loop-ligation method (Cost, 2007). In brief, the gene expression cassette is directly cut out from the respective parental plasmid. 5’ phosphorylated loop-forming oligonucleotides designed following the protocol by Cost are then ligated to form the dumbbell-shaped structure. An illustration of the production process is shown in Figure 2.


      Figure 2. Illustration of the production process for dumbbell vectors using the ELAN loop-ligation method. In this strategy, a parental plasmid containing the transgene expression cassette is first digested with two restriction endonucleases (BamHI and XhoI in this example) that recognize sites flanking the transcription unit. Next, 5’ phosphorylated oligonucleotides (ODNs) forming loop-structures with compatible restriction overhangs are ligated in the presence of the four restriction enzymes BglII, BamHI, XhoI and SalI which cleave homodimers formed by the loop ODN or the transcription unit. This strategy assists the generation of the correctly ligated dumbbell vector. Non-ligated sequences are subsequently destroyed by T7 DNA polymerase treatment, which exhibits strong 3’-5’ exonuclease activity (Engler and Richardson, 1983). RE, restriction enzyme.

    2. For the ELAN ligation reaction, loop ODN is added in a 50-fold molar excess over the linear expression unit DNA. Misligated by-products such as dimers formed of the loops or the transcription unit are cleaved by a set of four restriction enzymes (BamHI, BglII, XhoI, SalI) to foster dumbbell formation. A fifth restriction enzyme (AseI) is added to cleave dumbbells formed by ligation of the loop ODN with the plasmid backbone. This fifth enzyme can be a single or ideally a multi-cutter with respect to the cloning vector backbone but must not cut within the dumbbell vector. Non-ligated sequences are removed by exonuclease treatment. Detailed setups of the reactions are summarized in Table 2.

      Table 2. Three-step reaction procedure for the generation of luciferase-expressing dumbbells using the ELAN loop-ligation strategy. Step 1: Excision of the expression cassette from the respective parental plasmid, in this example using the enzymes XhoI and BamHI. Step 2: ELAN reaction including the T4 DNA ligase and five restriction endonuclease, in this example BglII, SalI, BamHI, XhoI, and AseI. Step 3: Exonuclease reaction using the T7 DNA polymerase. It is recommended to add again the restriction endonucleases of step 2 to ensure that any dumbbell-shaped byproducts taken over from step 2 are cleaved and rendered amendable for exonuclease degradation. After steps 1 and 2, enzymes are heat-inactivated but no further purification is needed.


    3. Quantity and quality of dumbbell vectors is analyzed by agarose gel electrophoresis as shown in Figure 3. Prior to the transfection of cells, dumbbell DNA is purified using the QIAquick PCR purification kit (QIAGEN) using the manufacturer’s instructions followed by ethanol precipitation. For the ethanol precipitation, the elution volume of 50 µl is topped up with distilled water to 400 µl. Then 40 µl of 3 M potassium acetate (pH 5.0) and 2.5 volumes (1,100 µl) of absolute ethanol are added, the solution is mixed by inverting the tube and placed for precipitation at -20 °C for 20 min. Vector DNA is pelleted by centrifugation at 16,100 x g for 15 min. The pellets are washed with 500 µl 4 °C 70% ethanol and air-dried. Purified DNA is then dissolved in TE buffer or distilled water.


      Figure 3. Dumbbell vectors for enhanced gene expression. Left side. Design of advanced dumbbell vectors: db-luc: basic vector; db-luc-enh: dumbbell featured with the SV40 enhancer; db-int-luc: dumbbell featured with an intron; db-int-luc-enh: dumbbell featured with SV40 enhancer and intron. Right side. Analytical 0.8% agarose gel electrophoresis of the four luciferase dbs after exonuclease treatment. The expected dumbbell bands but no by-products are detected. Numbers of lanes on the right correspond to the dumbbell numbers on the left. M: GeneRuler DNA Ladder Mix (Thermo Fisher Scientific).

  3. Transfection of human tissue culture cells with luciferase-expressing plasmids and dumbbells
    1. HEK293T and HepG2 cells are cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Hyclone) and 1% penicillin-streptomycin solution (Invitrogen). Cells are kept in humidified incubator with 5% CO2, and are passaged at 80-90% confluency.
    2. Cells are transfected using Lipofectamine 2000 (Invitrogen) following the manufacturer’s protocol. In brief, cells are seeded in 24-well plates one day prior to transfection at 70-80% confluency. At the day of transfection, 400 ng DNA is mixed with 1 µl Lipofectamine 2000 in Opti-MEM (Invitrogen) using a total volume of 100 µl. The DNA-liposome complexes are formed at room temperature during a 20 min incubation time, and then the mixture is added dropwise to the cells.
    3. Transfected cells are harvested and analyzed 48 h post transfection.

  4. Luciferase assays
    Measuring firefly luciferase enzyme activity in cell lysates using the luciferase assay system from Promega:
    1. For cell lysis, tissue culture medium is removed and 100 µl of passive lysis buffer (PLB, provided by the manufacturer) is added to each 24-well. Cells are incubated for 20 min at room temperature applying gentle shaking.
    2. After the lysis procedure, cell lysates are aliquoted, immediately frozen in liquid nitrogen, and stored at -80 °C. Though not further specified by the provider, frozen samples are stable for months or years.
    3. For the luciferase assay, 10 µl of each sample is transferred into an opaque white 96-well plate (Nunc).
    4. The Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek) system is used to inject Luciferase Assay Reagent (LARII) automatically and read the luminescence. Background signals are determined by blank and water controls for each assay and luciferase activity is normalized according to recommendations from Promega (Schagat et al., 2007).
    5. Representative data are shown in Figure 4.


      Figure 4. Enhancement of dumbbell vs. plasmid-driven luciferase expression by the β-globin gene chimeric intron and/or the full length SV40 enhancer. 5 x 104 HEK293T or HepG2 cells seeded in 24-wells were transfected with 400 ng dumbbell vectors or equivalent amounts of parental plasmids. Luciferase expression triggered by equimass amounts of dumbbell vectors and plasmids measured 48 h post transfection. Error bars indicate mean deviations from average of three to five independent experiments. Re: Relative.

Data analysis

Prism 5.0 GraphPad software is used for the data presentation and statistical analysis. Luciferase assay results are shown as mean ± SEM. For the comparison of the data, one-way ANOVA with Newman-Keuls post hoc test is used. * represents P value < 0.05, ** represents P value < 0.01, and *** represents P value < 0.001.

Notes

  1. 1 FD Unit is defined as 1 µl FastDigest enzyme by Thermo Fisher Scientific.
  2. According to our experience, the ligation reaction is completed within 4 h and longer incubation times do not improve the dumbbell yield.
  3. AseI is chosen to destroy the bacterial pGL3-Control plasmid backbone because it does not cleave within the dumbbell vector sequences comprising the gene of interest, the SV40 enhancer, and the intron. The choice of this enzyme however depends on the respective sequences of the dumbbell and the cloning vector backbone.
  4. T7 DNA polymerase exhibits 100% activity in the FD buffer and is therefore directly added into the ligation mixture.

Recipes

  1. 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. 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.
  2. Dean, D. A. (1997). Import of plasmid DNA into the nucleus is sequence specific. Exp Cell Res 230(2): 293-302.
  3. Dean, D. A., Dean, B. S., Muller, S. and Smith, L. C. (1999). Sequence requirements for plasmid nuclear import. Exp Cell Res 253(2): 713-722.
  4. Engler, M. J. and Richardson, C. C. (1983). Bacteriophage T7 DNA replication. Synthesis of lagging strands in a reconstituted system using purified proteins. J Biol Chem 258(18): 11197-11205.
  5. Hardee, C. L., Arevalo-Soliz, L. M., Hornstein, B. D. and Zechiedrich, L. (2017). Advances in non-viral DNA vectors for gene therapy. Genes (Basel) 8(2).
  6. Jiang, X. and Patzel, V. (2017). Formation of minimized hairpin template-transcribing dumbbell vectors for small RNA expression. Bio Protoc 7(11): e2313.
  7. 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.
  8. Luo, M and Reed, R (1999). Splicing is required for rapid and efficient mRNA export in metazoans. Proc Natl Acad Sci USA 96: 14937-14942.
  9. Schagat, T., Paguio, A. and Kopish, K. (2007). Normalizing genetic reporter assays: approaches and considerations for increasing consistency and statistical significance. Cell Notes 9-12.
  10. Schirmbeck, R., Konig-Merediz, S. A., Riedl, P., Kwissa, M., Sack, F., Schroff, M., Junghans, C., Reimann, J. and Wittig, B. (2001). Priming of immune responses to hepatitis B surface antigen with minimal DNA expression constructs modified with a nuclear localization signal peptide. J Mol Med (Berl) 79(5-6): 343-350.

简介

唯一包含治疗相关序列的最小DNA载体对于新型治疗方案的发展具有很大的希望。哑铃型载体代表已经进入临床发展的晚期阶段的非病毒非整合DNA最小载体(Hardee等人,2017)。可接合的内含子和DNA核输入信号如SV40增强子序列是在质粒载体中发现多个应用以改善转基因表达的分子特征。然而,在哑铃中,由内含子引发的效应未被研究,基于DNA的核导入序列尚未发现应用,可能是因为哑铃载体在尺寸上不断被最小化。我们调查内含子和/或SV40增强子衍生序列对哑铃载体驱动的报告基因表达的影响。发现可拼接内含子的实现在所有研究的细胞系中无条件地增强基因表达。相反,SV40增强子的使用改善了细胞类型依赖性的基因表达。虽然这两个特征显着增加哑铃载体大小,但内含子和增强子或两者的组合都不会对基因表达产生负面影响。相反,与质粒或对照哑铃相比,这两个特征在一起改善了哑铃驱动的基因表达,高达160-或56倍。因此,强烈建议考虑用于哑铃矢量设计的内含子和SV40增强子。这种先进的设计可以促进哑铃型DNA载体的临床前和临床应用。
【背景】虽然许多基因已经使用哑铃形DNA载体表达,但大多数这些应用使用包括启动子,编码序列(CDS)和转录终止子的基本设计。一些载体包括嵌合内含子,然而,没有报道通过这种设计增强了转基因表达(Schirmbeck等人,2001)。在这里,我们研究了分子特征引发的效应,这些特征经常在哑铃驱动基因表达的质粒设计中得到应用:1.已知来自人β-珠蛋白基因剪接的嵌合内含子促进RNA加工,核出口,随后基因表达(Luo and Reed,1999);和猿猴病毒40(SV40)增强子,其可以增强同源SV40启动子的活性,并且另外据报道其作为活性DNA核进口序列起作用(Dean,1997; Dean等人, ,1999)。提出的这一现象背后的机制是,SV40增强剂在蛋白质核进口机制的支持下招募了在细胞质中携带蛋白质核进入信号的转录因子,载体DNA携带载体转移到细胞核中。我们生成了含有荧光素酶的哑铃载体,只含有一个或没有这些分子特征,并监测HEK293T和HepG2细胞中的转基因表达。我们的数据表明,内含子和SV40增强子可以大大改善哑铃矢量设计(Jiang等人,2016)。

关键字:哑铃形载体, 最小DNA载体, 转基因表达, 基因疗法, 内含子, SV40增强子

材料和试剂

  1. 移液针头(Corning,supo)®/ sup> Isotip ®过滤0.2-10μl)(Corning,目录号:4807)
  2. 移液头(Axygen ® TF200RS1-200μl)(Corning,Axygen ,目录号:TF-200-R-S)
  3. 移液器吸头(Axygen ® TF1000RS100-1,000μl)(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 TM 将50ml圆锥形离心管(Corning,Falcon ®,目录号:352070)
  7. T-75烧瓶(Corning,目录号:3290)
  8. 24孔细胞培养板(Corning,目录号:3527)
  9. 96孔板(Thermo Fisher Scientific,Thermo Scientific TM,目录号:442404)
  10. HEK293T细胞(ATCC,目录号:CRL-3216)
  11. HepG2细胞(ATCC,目录号:HB-8065)
  12. pGL3对照载体(Promega,目录号:E1741)
  13. 单击® TOP10化学能力 E。大肠杆菌(Thermo Fisher Scientific,Invitrogen TM,目录号:C404010)
  14. 嵌合人β-珠蛋白内含子序列(Gene synthesis,GeneArt,Applied Biosystems):5'-CAGGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACAGAGACGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGG-3'
  15. SV40增强子序列:5'-CGATGGAGCGGAGAATGGGCGGAACTGGGCGGAGTTA GGGGCGGGATGGGCGGAGTTAGGGGCGGGACTATGGTTGCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGCTGGGGAGCCTGGGGACTTTCCACACCTGGTTGCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGCTGGGGAGCCTGGGGACTTTCCACACCCTAACTGACACACATTCCACAGC-3’
  16. 用于嵌合内含子扩增的寡核苷酸引物(综合DNA技术合成,25nmol,去保护脱盐):
    内含子-Fw:5'-ATCTATCGGGATCCAAGCTTCAGGTAAGTATCAAGGTTACAAGACAGG-3'
    内含子-Rv:5'-CATTATCTGGATCCCCATGGACCCTGTGGAGAGAAAGGCAA-3'
  17. 循环序列(综合DNA技术合成,25nmol,脱保护脱盐):
    环-1:5'-pGATCTGACCAGTTTTCTGGTCA-3'
    环2:5'-pTCGACAGGCTCTTTTGAGCCTG-3'
  18. 聚(A)信号的寡核苷酸引物(由Integrated DNA Technologies合成,25nmol级,去保护脱盐):
    polyA-Fw:5'-TGTAATTCTAGAGTCGGGGCG-3'
    polyA-Rv:5'-ATCTATCGGGATCCTTACCACATTTGTAGAGGTT-3'
  19. FastDigest III(Thermo Fisher Scientific,Thermo Scientific TM,目录号:FD0504)
  20. FastDigest I(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:FD0573)
  21. FastDigest Bam(Thermo Fisher Scientific,Thermo Scientific TM,目录号:FD0054)
  22. FastDigest II(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:FD0083)
  23. FastDigest Xho I(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:FD0694)
  24. FastDigest I(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:FD0644)
  25. FastDigest I(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:FD0914)
  26. UltraPure TM无DNA酶/无RNA酶的蒸馏水(Thermo Fisher Scientific,Invitrogen TM,目录号:10977015)
  27. T7 DNA聚合酶(10U /μl)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:EP0081)
  28. DNA聚合酶,重组体(5U /μl)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:EP0402)
  29. 10x FD缓冲液(Thermo Fisher Scientific,Thermo Scientific TM,目录号:B64)
  30. T4 DNA连接酶(5U /μl)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:EL0014)
  31. dNTP设置100mM溶液(Thermo Fisher Scientific,Thermo Scientific TM,目录号:R0181)
  32. QIAquick PCR纯化试剂盒(QIAGEN,目录号:28106)
  33. 醋酸钾(Sigma-Aldrich,目录号:P1190-100G)
  34. 琼脂糖,LE,分析纯(Promega,目录号:V3125)
  35. 乙醇,绝对(Fisher Scientific,目录号:BP28184)
  36. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S9888-500G)
  37. 六氯化镁(MgCl 2·6H 2 O)(Sigma-Aldrich,目录号:M2670-100G)
  38. Tris-HCl(Roche Diagnostics,目录号:10812846001)
  39. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:EDS-100G)
  40. 腺苷5'-三磷酸二钠盐水合物(Sigma-Aldrich,目录号:A2383-1G)
  41. 溴化乙锭溶液(Bio-Rad Laboratories,目录号:1610433)
  42. 苯酚溶液(Sigma-Aldrich,目录号:P4557-100ML)
  43. 氯仿(Sigma-Aldrich,目录号:288306-1L)
  44. 3-甲基-1-丁醇(Sigma-Aldrich,目录号:309435-100ML)
  45. GeneRuler DNA梯形混合物(Thermo Fisher Scientific,Thermo Scientific TM,目录号:SM0331)
  46. HyClone Dulbecco的改良Eagles中/高葡萄糖与L-谷氨酰胺,丙酮酸钠(GE Healthcare,HyClone TM,目录号:SH30243.01)
  47. HyClone标准胎牛血清(GE Healthcare,HyClone TM,目录号:SH30088.03)
  48. 青霉素 - 链霉素(10,000U / ml)(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  49. 转染试剂(Thermo Fisher Scientific,Invitrogen TM,目录号:11668019)
  50. Opti-MEM ® I还原血清培养基(Thermo Fisher Scientific,Gibco TM,目录号:31985070)
  51. 荧光素酶测定系统(Promega,目录号:E1501)
  52. 液氮
  53. TE缓冲(见配方)

设备

  1. 移液器(Gilson,PIPETMAN Classic ,P2,P20N,P200N和P1000N)
  2. CO 2培养箱(Thermo Electron)
  3. 振荡器(Heidolph Instruments,型号:Unimax 2010)
  4. 玻璃烧杯(Schott,Duran)
  5. 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。
  6. Gel doc(Bio-Rad Laboratories,Gel Doc Imager)
  7. 凝胶运行装置(Amersham Biosciences)
  8. 凝胶染色托盘(GE Healthcare)
  9. 台式离心机(Eppendorf,型号:5430 R)
  10. 热块(Thermomixer舒适)(Eppendorf)
  11. 分光光度计(Thermo Fisher Scientific,Thermo Scientific TM,型号:NanoDrop TM 2000)
  12. 微波(松下)
  13. II级生物安全柜(Gelman)
  14. Synergy H1多模式阅读器(BioTek Instruments,型号:Synergy H1)

软件

  1. GraphPad Prism软件5.0

程序

  1. 荧光素酶表达质粒变体的设计和分子克隆
    为了研究合成内含子和/或SV40增强子对哑铃驱动基因表达的功能,我们基于pGL3对照载体克隆相应的质粒载体。 pGL3对照载体在SV40启动子的控制下携带萤火虫荧光素酶基因以及表达盒的3'末端的SV40增强子元件。克隆如下所述进行:
    1. 通过基因合成(GeneArt,Applied Biosystems)合成137bp嵌合人β-珠蛋白微内含子,使用引物内含子-Fw和内含子-Rv进行PCR扩增,然后使用HindIII插入到pGL3-对照质粒III和Nco I位点以创建内含子和SV40增强子(int-luc-enh)(Jiang等人)特有的荧光素酶表达载体,2016和2017)。生产过程的说明如图1所示

      图1.质粒克隆方案的图示。 步骤1:质粒p-int-luc-enh是通过将b-珠蛋白微型内含子插入到HindⅢ和NcoI区的载体pGL3-Control(p-luc-enh)。步骤2:通过SV40增强子与载体pGL3-Control(p-luc-enh)的SV40聚(A)位点通过SV40 polyA位点使用Xba 进行交换,可以产生质粒p-luc我和 Bam HI。步骤3:通过用SV40 polyA位点将SV40增强子与载体p-int-luc-enh的SV40 polyA位点交换,使用Xba I和Bam HI。

    2. pGL3对照中的SV40增强子序列通过用XbaI和BamHI HI消化载体而被删除。通过这样做,我们也删除了SV40晚期聚(A)信号。为了检索SV40聚(A)信号,使用引入了Xba I和Bam HI位点的引物polyA-Fw和polyA-Rv对其序列进行PCR扩增。用XbaI和Bam HI切割PCR产物,并重新插入SV40 poly(A)信号以产生缺少内含子和SV40增强子的质粒(luc)(Jiang等人,2016和2017)。生产过程的说明如图1所示
    3. 从含有内含子和SV40增强子的pGL3对照变体开始重复步骤A2中描述的等同过程,以产生仅具有内含子(int-luc)(Jiang等人)的质粒>,2016)。生产过程的说明如图1所示
    4. 为了简化,将新名称分配给表1所列的结构。

      表1.荧光素酶表达构建体的命名。 质粒被分配前缀'p',哑铃前缀'db'。


  2. 生产荧光素酶表达的哑铃载体
    1. 使用由核酸酶(ELAN)循环连接法(Cost,2007)辅助的酶连接,由方法A所述的相应质粒产生荧光素酶表达的哑铃载体。简而言之,从相应的亲本质粒直接切出基因表达盒。然后将按“费用”方案设计的5'磷酸化环形成寡核苷酸结合形成哑铃形结构。生产过程的说明如图2所示

      图2.使用ELAN循环连接方法的哑铃向量生产过程的图示。 在该策略中,首先用识别位点的两个限制性内切核酸酶(本例中为BamHI和Xho I)消化含有转基因表达盒的亲本质粒侧翼转录单位。接下来,在四种限制性内切酶BglII,BamHI,HindⅢ和BamHI的存在下连接形成具有相容限制突出端的环结构的5'磷酸化寡核苷酸(ODN) Xho I和Sal I,其切割由环ODN或转录单元形成的同源二聚体。该策略有助于生成正确连接的哑铃矢量。随后通过T7 DNA聚合酶处理破坏非连接序列,其显示出强的3'-5'核酸外切酶活性(Engler和Richardson,1983)。 RE,限制酶
    2. 对于ELAN连接反应,在线性表达单元DNA上以50倍摩尔过量加入环ODN。由循环或转录单元形成的二聚体等错配的副产物被一组四种限制性内切酶切割(BamHI,BglII,Xho, / em> I, Sal I)以促进哑铃形成。加入第五限制性内切酶(Ise I)以切割通过连接ODN与质粒骨架形成的哑铃。该第五种酶可以是相对于克隆载体骨架的单个或理想的多切割机,但不能在哑铃载体内切割。通过外切核酸酶处理除去未连接的序列。反应的详细设置总结在表2中。

      表2.使用ELAN循环连接策略产生荧光素酶表达哑铃的三步反应程序。步骤1:从相应的亲本质粒切除表达盒,在该实施例中使用酶Xho I和Bam HI。步骤2:包括T4DNA连接酶和5个限制性内切核酸酶的ELAN反应,在该实施例中,Bgl II,SalI,Bam HI, > Xho I和 Ase I。步骤3:使用T7 DNA聚合酶进行核酸外切酶反应。建议再次添加步骤2的限制性内切核酸酶,以确保从步骤2接管的任何哑铃形副产物被切割并使其可修饰以用于外切核酸酶降解。在步骤1和2之后,酶被热灭活,但不需要进一步纯化。


    3. 通过琼脂糖凝胶电泳分析哑铃载体的数量和质量,如图3所示。在转染细胞之前,使用制造商的说明书使用QIAquick PCR纯化试剂盒(QIAGEN)纯化哑铃DNA,然后乙醇沉淀。对于乙醇沉淀,将50μl的洗脱体积用蒸馏水加满至400μl。然后加入40μl3M乙酸钾(pH5.0)和2.5体积(1100μl)无水乙醇,通过倒置管将该溶液混合并置于-20℃沉淀20分钟。通过以16,100×g离心15分钟将载体DNA沉淀。颗粒用500μl4℃70%乙醇洗涤并风干。纯化的DNA然后溶于TE缓冲液或蒸馏水中

      图3.用于增强基因表达的哑铃载体。左侧。高级哑铃载体设计:db-luc:基本载体; db-luc-enh:具有SV40增强子功能的哑铃; db-int-luc:哑铃特色的内含子; db-int-luc-enh:具有SV40增强子和内含子的哑铃。右边。核酸外切酶处理后四种荧光素酶dbs的0.8%琼脂糖凝胶电泳分析。检测到预期的哑铃带,但没有副产物。右边的车道数量对应于左边的哑铃号码。 M:GeneRuler DNA梯形混合物(Thermo Fisher Scientific)。

  3. 用荧光素酶表达质粒和哑铃转染人组织培养细胞
    1. 将HEK293T和HepG2细胞在补充有10%(v / v)热灭活的胎牛血清(Hyclone)和1%青霉素 - 链霉素溶液(Invitrogen)的Dulbecco改良的Eagle培养基(DMEM,Invitrogen)中培养。将细胞保持在5%CO 2的湿润培养箱中,并以80-90%汇合流通。
    2. 使用Lipofectamine 2000(Invitrogen)按照制造商的方案转染细胞。简言之,在转染前一天将细胞接种在24孔板中,以70-80%汇合。在转染当天,将400ng DNA与Opti-MEM(Invitrogen)中的1μlLipofectamine 2000混合,总体积为100μl。在20分钟孵育时间内,室温下形成DNA-脂质体复合物,然后将混合物滴加到细胞中。
    3. 收获转染的细胞并在转染后48小时进行分析
  4. 萤光素酶测定
    使用Promega的荧光素酶测定系统测量细胞裂解物中萤火虫荧光素酶的活性:
    1. 对于细胞裂解,去除组织培养基,并向每个24孔加入100μl被动裂解缓冲液(PLB,由制造商提供)。细胞在室温下温和摇匀20分钟
    2. 裂解程序后,将细胞裂解物等分,立即在液氮中冷冻,并储存在-80℃。虽然供应商没有进一步规定,但冻结样品稳定了数月或数年。
    3. 对于荧光素酶测定,将10μl每个样品转移到不透明的白色96孔板(Nunc)中。
    4. Synergy H1混合多模式微孔板读数器(BioTek)系统用于自动注入萤光素酶测定试剂(LARII)并读取发光。背景信号由每个测定的空白和水对照确定,荧光素酶活性根据Promega(Schagat等人,2007)的推荐标准化。
    5. 代表性数据如图4所示

      图4.通过β-珠蛋白基因嵌合内含子和/或全长SV40增强子增强哑铃与质粒驱动的荧光素酶表达 5×10 4 HEK293T或用400ng哑铃载体或等量的亲本质粒转染接种在24孔中的HepG2细胞。在转染后48小时测量由等量量的哑铃载体和质粒触发的荧光素酶表达。误差条表示从三到五个独立实验的平均值的平均偏差。 Re:相对

数据分析

Prism 5.0 GraphPad软件用于数据呈现和统计分析。荧光素酶测定结果显示为平均值±SEM。为了比较数据,使用单因素ANOVA与Newman-Keuls事后检验。 *表示P 值< 0.05,**表示P 值< 0.01,***表示P 值< 0.001。

笔记

  1. 1 FD单位由Thermo Fisher Scientific定义为1μlFastDigest酶。
  2. 根据我们的经验,连接反应在4小时内完成,较长的孵育时间不能提高哑铃产量。
  3. 我选择破坏细菌pGL3-Control质粒骨架,因为它不包含目标基因,SV40增强子和内含子的哑铃载体序列内切割。然而,该酶的选择取决于哑铃和克隆载体骨架的相应序列
  4. 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

食谱

  1. TE缓冲区
    10mM Tris-HCl,pH8.0
    1 mM EDTA

致谢

这里描述的方案在以前在Jiang等开发和使用。 (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. 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。
  2. Dean,DA(1997)。&nbsp; 将质粒DNA导入细胞核具有序列特异性。 Exp Cell Res 230(2):293-302。
  3. Dean,DA,Dean,BS,Muller,S. and Smith,LC(1999)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/ 10585295“target =”_ blank“>质粒核导入的序列要求。 Exp Cell Res 253(2):713-722。
  4. Engler,MJ和Richardson,CC(1983)。&nbsp; 噬菌体T7 DNA复制。使用纯化的蛋白质在重构系统中合成滞后链。生物化学258(18):11197-11205。
  5. Hardee,CL,Arevalo-Soliz,LM,Hornstein,BD和Zechiedrich,L。(2017)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/ pubmed / 28208635“target =”_ blank“>用于基因治疗的非病毒DNA载体的进展基因(巴塞尔) 8(2)。
  6. Jiang,X.和Patzel,V.(2017)。形成最小化的发夹模板 - 转录哑铃载体用于小RNA表达。 Bio Protoc 7(11):e2313。
  7. 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。
  8. Luo,M和Reed,R(1999)。&nbsp; 为了在后生动物中快速有效地进行mRNA的输出,需要进行拼接。 Proc Natl Acad Sci USA 96:14937-14942。
  9. Schagat,T.,Paguio,A.和Kopish,K.(2007)。归一化遗传报告分析:增加一致性和统计学显着性的方法和注意事项。细胞注释 9-12。
  10. Schirmbeck,R.,Konig-Merediz,SA,Riedl,P.,Kwissa,M.,Sack,F.,Schroff,M.,Junghans,C.,Reimann,J.and Wittig,B。(2001) ; 以最小的DNA表达启动对乙型肝炎表面抗原的免疫应答 使用核定位信号肽修饰的构建体。J.Mol Med(Berl)79(5-6):343-350。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Jiang, X. and Patzel, V. (2017). Advanced Design of Minimalistic Dumbbell-shaped Gene Expression Vectors. Bio-protocol 7(15): e2425. DOI: 10.21769/BioProtoc.2425.
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