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Construction and Cloning of Minigenes for in vivo Analysis of Potential Splice Mutations
用于体内潜在的剪接突变分析的微小基因的构建及克隆   

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PLOS Genetics
Jan 2016

Abstract

Disease-associated mutations influencing mRNA splicing are referred to as splice mutations. The majority of splice mutations are found on exon-intron boundaries defining canonical donor and acceptor splice sites. However, mutations in the coding region (exonic mutations) can also affect mRNA splicing. Exact knowledge of the disease mechanism of splice mutations is essential for developing optimal treatment strategies. Given the large number of disease-associated mutations thus far identified, there is an unmet need for methods to systematically analyze the effects of pathogenic mutations on mRNA splicing. As splicing can vary between cell types, splice mutations need to be tested under native conditions if possible. A commonly used tool for the analysis of mRNA splicing is the construction of minigenes carrying exonic and intronic sequences. Here, we describe a protocol for the design and cloning of minigenes into recombinant adeno-associated virus (rAAV) vectors for gene delivery and investigation of mRNA splicing in a native context. This protocol was developed for minigene-based analysis of mRNA splicing in retinal cells, however, in principle it is applicable to any cell type, which can be transduced with rAAV vectors.

Keywords: Minigene (微小基因), mRNA splicing (mRNA剪接), Cloning (克隆), rAAV (腺相关病毒), Analysis of mutations (分析突变)

Background

A substantial portion of disease-associated mutations (at least 15%) are predicted to result in aberrant mRNA splicing (Cartegni et al., 2002; Singh and Cooper, 2012; Sterne-Weiler and Sanford, 2014). The ‘classical’ splice mutations are those affecting the canonical sequences defining the 5’- and 3’-splice sites (donor and acceptor splice sites, respectively). However, splice mutations can also occur in other non-coding and coding regions (Wang and Cooper, 2007; Scotti and Swanson, 2016). There is growing evidence that the frequency of splice mutations in coding regions (exonic mutations) has been underestimated (Julien et al., 2016; Soukarieh et al., 2016). Exonic splice mutations (i.e., point mutations, insertions or deletions) can induce exon skipping, intron retention or lead to the generation of novel donor or acceptor splice sites. Depending on the gene and exon composition, these mechanisms can have different impacts on the protein level ranging from haploinsufficiency to gain of function. Nevertheless, the exact knowledge of molecular mechanisms underpinning the disease-causing mutations is essential for developing optimal treatments.

mRNA splicing occurs in a highly cell type-specific manner, highlighting the need to analyze the impact of potential splice mutations in the tissue which is primarily affected by the mutation (Wang et al., 2008). Consequently, in the optimal case, mRNA splicing should be analyzed on the native gene and in the native tissue. This option, however, is rather demanding for several reasons:
1) It might require the elaborate generation of genetically modified human cell lines. This impedes a more systematic analysis of splice mutations for a single gene.
2) Many native cell types are highly specialized and their cultured counterparts (if available at all) do not reflect every morphological and molecular hallmark of the native cells including the composition and activity of the splicing machinery.
3) The generation of humanized animal models expressing the respective splice mutation in a given tissue is not only technically challenging, but also time-consuming and costly. Therefore, this approach also appears rather unsuitable for systematic testing of splice mutations for a given gene.
4) Often, native genes are too large to be cloned into classical expression vectors.

One alternative to circumvent a number of these obstacles is to use human minigenes designed for expression in appropriate animal models (e.g., mouse). We have evaluated this approach in recent studies addressing the effects of disease-associated mutations in different genes, e.g., PRPH2, on mRNA splicing (Becirovic et al., 2016b; Nguyen et al., 2016; Khan et al., 2017; Petersen-Jones et al., 2017). For stable and specific ectopic expression of the minigenes, we took advantage of rAAV vectors. These vectors are capable of transducing a variety of different cell types in vivo (Zincarelli et al., 2008; Lisowski et al., 2014). Furthermore, the design, cloning, production and purification of rAAV vectors can be completed in a few weeks and does not require elaborate technical equipment (Becirovic et al., 2016a).

Most native genes including PRPH2 exceed the limited packaging capacity of AAVs (approx. 4.7 kb) (Wu et al., 2010). Therefore, we designed PRPH2 minigenes lacking large intronic parts, which usually do not contain information required for correct mRNA splicing. For genes which do not contain large exon numbers or sizes, shortening of the intronic sections also allows for introducing the entire protein coding region into the rAAV vector-based minigenes.

This strategy (cf. Figure 1) was developed and evaluated to analyze the impact of known disease-linked mutations on mRNA splicing and protein expression in photoreceptor-specific genes, but should in principle also be transferable to other cell types.


Figure 1. Workflow schematic for construction and cloning of minigenes

Materials and Reagents

  1. Isolation of human genomic DNA for cloning of minigenes
    1. 1.5 ml Eppendorf tubes (SARSTEDT, catalog number: 72.690.001 )
    2. Scalpel (Swann Morton, catalog number: 0510 )
    3. Gentra Puregene Buccal Cell Kit (QIAGEN, catalog number: 158845 )
    4. 2-Propanol (100%, ACS, ISO, Reag. Ph. Eur. grade)
    5. Ethanol (70%, ACS, ISO, Reag. Ph. Eur. grade)

  2. Overlap extension PCR for construction and cloning of minigenes
    1. PCR tubes (BRAND, catalog numbers: 781320 and 781334 )
    2. Expression vector of choice (e.g., pcDNA3.1 vector (+), Thermo Fisher Scientific, InvitrogenTM, catalog number: V79020 )
    3. Double distilled H2O (ddH2O)
    4. Herculase II Fusion DNA Polymerase (Agilent Technologies, catalog number: 600675 )
    5. dNTPs, 10 mM (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0192 )
    6. DNA Loading Dye (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0611 )
    7. GeneRuler 1 kb Plus DNA Ladder (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: SM1331 )
    8. EDTA (VWR, catalog number: 20302.293 )
    9. Tris-(hydroxymethyl) aminomethane (VWR, catalog number: 103156X )
    10. Boric acid (VWR, catalog number: 20185.360 )
    11. QIAquick Gel Extraction Kit (QIAGEN, catalog number: 28704 )
    12. 1x TBE buffer (see Recipes)

  3. Site-directed mutagenesis to introduce point mutations of interest into minigene
    1. PCR tubes (BRAND, catalog numbers: 781320 and 781334 )
    2. QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, catalog number: 210518 )
    3. Double distilled H2O (ddH2O)
    4. Peptone (Applichem, catalog number: 403898.1210 )
    5. Yeast extract (Applichem, catalog number: A1552 )
    6. NaCl (VWR, catalog number: 27810.364 )
    7. D-(+)-Glucose (Sigma-Aldrich, catalog number: 49159 )
    8. Agar (Sigma-Aldrich, catalog number: A5054 )
    9. Appropriate antibiotic for the plasmid vector (e.g., Ampicillin, Carl Roth, catalog number: K029.2 )
    10. LB agar plates containing the appropriate antibiotic for the plasmid vector (see Recipes)
    11. LB medium (see Recipes)

  4. Subcloning of minigenes into rAAV vector
    1. Appropriate rAAV cis vector plasmid (e.g., pAAV-MCS2, Addgene, catalog number: 46954 )

Equipment

  1. Pipettes (e.g., Eppendorf)
  2. Vortexer (Heidolph Instruments, model: Reax top )
  3. Incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: Heraeus B12 Function Line, catalog number: 50042307 )
  4. Microcentrifuge (Eppendorf, model: Centrifuge MiniSpin® , catalog number: 5452000018)
  5. Two water baths (Haake, catalog number: 003-2859 )
  6. Thermocycler (Thermo Fisher Scientific, model: ProFlexTM, catalog number: 4483636 )
  7. NanoDropTM (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: ND-2000C )

Software

  1. Human splicing finder software (e.g., http://www.umd.be/HSF3/)
  2. Tm Calculator Software (e.g., NEB Tm Calculator)

Procedure

  1. In silico splice analysis
    In many cases, initial in silico prediction of mRNA splicing of the single mutations might be helpful. To predict the potential effects on splicing of your mutation of interest use a human splicing finder software (e.g., http://www.umd.be/HSF3/).
    1. Go to http://www.umd.be/HSF3/ and select an analysis type. We recommend using the option ‘Analyze a sequence’.
    2. Choose a sequence. We recommend using the option ‘Pasting your own sequence’. Paste the wildtype version of your sequence of interest into the provided box and click on ‘Proceed to analysis’.
      Note: Your sequence should contain at least 40 bp flanking the respective point mutation on both sides.
    3. Click on ‘Perform a quick mutation’, type in the position of your mutation of interest relative to the sequence you have entered before and select the new nucleotide. Then click on ‘Proceed to quick mutation analysis’.
    4. Human splice finder webserver output:
      1. Click on ‘Raw Data Tables’ to analyze the effect of the entered point mutation in detail.
      2. You are provided with five different data tables called ‘Potential splice sites’, ‘Potential Branch Points’, ‘Enhancer motifs’, ‘Silencer motifs’ and ‘Other splicing motifs’. Each of them shows the percental variation induced by the point mutation. Newly formed splice sites or motifs are indicated in green, a change in strength of an existing site or motif is indicated in light yellow.
    5. Repeat the analysis for all point mutations of interest and select the ones predicted to affect splicing for further cloning.

  2. Isolation of human genomic DNA for cloning of minigenes
    1. Preheat one water bath to 65 °C and the other to 37 °C.
    2. To collect buccal cells, scrape the inside of the mouth 10 times with a Buccal Collection Brush (provided in the kit, Materials and Reagents A3).
      Note: For best results, wait at least 1 h after eating or drinking to collect buccal cells.
    3. Dispense 300 µl Cell Lysis Solution (provided in the kit, Materials and Reagents A3) into a 1.5 ml Eppendorf tube. Remove the Collection brush from its handle using a sterile scalpel, and place the detached head in the tube.
    4. Incubate at 65 °C for 60 min to complete cell lysis.
    5. Remove the collection brush head from the Cell Lysis Solution, scraping it on the side of the tube to recover as much liquid as possible.
    6. Add 1.5 µl RNase A Solution (provided in the kit), and mix by inverting the tube 25 times.
    7. Incubate for 30 min at 37 °C. Incubate for 1 min on ice to cool the sample.
    8. Add 100 µl Protein Precipitation Solution (provided in the kit, Materials and Reagents A3), and vortex vigorously for 20 sec at high speed.
    9. Incubate for 5 min on ice.
    10. Centrifuge for 3 min at 13,000-16,000 x g to pellet the precipitated proteins.
    11. Pipet 300 µl isopropanol and 0.5 µl Glycogen Solution (provided in the kit, Materials and Reagents A3) into a clean 1.5 ml Eppendorf tube, and add the supernatant from the previous step by pouring or pipetting carefully. Make sure not to transfer the protein pellet.
    12. Mix by inverting the tube gently 50 times or by rotating it for 2.5 min at 20 rpm.
    13. Centrifuge at RT for 5 min at 13,000-16,000 x g.
    14. Carefully discard the supernatant, and drain the tube by inverting it on a clean piece of absorbent paper, taking care that the pellet remains in the tube.
    15. Add 300 µl of 70% ethanol and invert the tube several times to wash the DNA pellet.
    16. Centrifuge at RT for 1 min at 13,000-16,000 x g.
    17. Carefully discard the supernatant. Drain the tube on a clean piece of absorbent paper, taking care that the pellet remains in the tube. Allow to air dry for 5 min.
    18. Add 100 µl DNA Hydration Solution (provided in the kit, Materials and Reagents A3) and vortex for 5 sec at medium speed to mix.
    19. Incubate at 65 °C for 1 h to dissolve the DNA.
    20. Incubate at room temperature overnight with gentle shaking. Ensure tube cap is tightly closed to avoid leakage. Samples can then be centrifuged briefly and transferred to a storage tube.

  3. Overlap extension PCR for construction and cloning of minigenes
    C1. Methodological description and design of overlap primers
    To enable minigene-based analysis of mutations on mRNA splicing, large introns can be shortened. In our recent studies, we validated that approx. 200 bp of the intronic regions flanking the exons are sufficient for correct mRNA splicing (Becirovic et al., 2016b; Nguyen et al., 2016).
    To generate the appropriate minigenes via standard cloning procedures, an overlap extension PCR can be used. For this purpose, as a first step the respective exons including ≥ 200 bp of the flanking introns must be amplified via standard PCR. The primers used for this PCRs must encompass defined overhangs of approx. 20 bp. These overhangs are complementary to the corresponding PCR fragment to be connected in the second overlap extension PCR. These create 40 bp overlap regions used for the amplification of the two fragments. We applied this strategy for creating minigenes consisting of three native exons including two shortened flanking introns (Figure 2). However, this method is also suitable for the generation of minigenes with a random number of exons and shortened introns.
    1. Design PCR primers to amplify one exon plus ≥ 200 bp of the flanking intronic regions. Check the melting temperature (Tm) using a Tm Calculator Software (e.g., NEB Tm Calculator).
    2. To create the overlap regions, the primer must contain the approx. 20 bp long region complementary to the adjoining PCR amplicon (see Figure 2).
      Example: When designing primers to amplify the second exon plus flanking intronic regions (amplicon #2), add the sequence of the final 20 bp in the exon 1 containing amplicon (amplicon #1) to the 5’ end of your forward primer. Next, add the reverse sequence of the first 20 bp of the exon 3 containing amplicon (amplicon #3) to the 5’ end of your reverse primer. These overlapping sequences will be needed for subsequent overlap extension PCRs.


      Figure 2. Schematic of overlap extension PCR for creating minigenes consisting of three exons and two flanking introns that were shortened. The single amplicons are depicted in different colors. Restriction sites are shown in blue. Primers are depicted bicolored according to their templates and matching overlaps. Two PCR amplicons from step 1 serve as templates for the overlap PCR 1 and can be combined together in a random manner (e.g., amplicons of PCR 1 + PCR 2). The final overlap PCR 2 can be performed with the remaining PCR fragment (in this example, the amplicon of PCR 3). For all overlap PCRs, only the 5’ primer of the first and the 3’ primer of the second PCR amplicon should be used.

    3. For the amplification of the first minigene exon: Add an appropriate restriction site to the 5’ end of your forward primer according to the restriction site of your expression vector (e.g., HindIII in pcDNA3.1). Consider adding an additional sequence (up to 4 bp) to the 5’ end of the restriction site for more efficient cleavage by the restriction enzymes.
      For amplification of the last minigene exon: Add an appropriate restriction site to the 5’ end of your reverse primer according to the restriction site of your expression vector (e.g., NotI). Consider adding additional up to 4 bp to the 5’ end of the restriction site for more efficient cleavage by the restriction enzymes.
    4. Order PCR primers (approx. 0.05 µmol, at least HPSF purified) from commercial providers (e.g., Eurofins Genomics).

    C2. PCR amplification of full-length exons and shortened introns
    1. To amplify the single exon and flanking intronic regions, prepare the sample reaction as follows:
      X µl
      Genomic DNA (100-300 ng)
      5 µl
      5x Herculase II Fusion buffer*
      1 µl
      dNTPs (10 mM)
      1 µl
      Forward primer (10 µM)
      1 µl
      Reverse primer (10 µM)
      0.25 µl
      Herculase II Fusion DNA Polymerase*
      Add to 25 µl
      Double distilled H2O
      *Note: Provided in the kit (Materials and Reagents B4).
    2. Mix all reagents in a PCR tube by pipetting up and down.
    3. For the PCR use the following cycling parameters:

      Note: For optimal results the annealing temperature can be adapted as required.
    4. After Amplification, add 5 µl 6x DNA Loading Dye to your PCR reaction, load it on a 0.7% agarose gel together with a DNA standard of your choice (e.g., GeneRuler 1 kb Plus DNA Ladder) and run it in 1x TBE buffer. Check for correct amplification.
    5. If amplification was successful, i.e., if you see a band of correct size, excise it using a scalpel and put the gel slice into an Eppendorf tube. Purify DNA using the gel extraction kit according to the manufacturer’s instructions.
    6. Measure the DNA concentration using NanoDrop.
    7. Repeat Step C2 for all exons.

    C3. Overlap extension PCR
    1. To join the first two amplicons containing exon 1 and 2, prepare the sample reaction as follows:
      X µl
      Amplicon #1 (100-150 ng)
      X µl
      Amplicon #2 (use molar ratio 1:1)
      5 µl
      5x Herculase II Fusion buffer
      1 µl
      dNTPs (10 mM)
      1 µl
      Forward primer of Amplicon #1 (10 µM)
      1 µl
      Reverse primer of Amplicon #2 (10 µM)
      0.25 µl
      Herculase II Fusion DNA Polymerase
      Add to 25 µl
      Double distilled H2O
    2. Mix all reagents in a PCR tube by pipetting up and down.
    3. For the PCR use the following cycling parameters:

      Note: For optimal results the annealing temperature can be adapted as necessary.
    4. Load all of your PCR reaction on an agarose gel and check for correct amplification.
    5. If amplification was successful, i.e., if you see a band of correct size, excise the band and purify DNA using the gel extraction kit according to the manufacturer’s instructions.
    6. Measure the DNA concentration using NanoDrop.
    7. Repeat Overlap extension PCR to connect the new amplicon with the subsequent amplicon. For this, use the purified DNA of both amplicons as template; and the forward primer of amplicon #1 and the reverse primer of amplicon #3 as Overlap extension PCR primers.
    8. Depending on the number of amplicons to be joined together, Steps 1-7 can be repeated.
    9. Use the chosen restriction sites to clone your minigene into the respective expression vector using standard cloning techniques.
    10. Check for the correct sequence of the minigene by sequencing (e.g., Eurofins Genomics).
      Notes:
      1. Consider using an expression vector carrying a reporter gene (e.g., GFP) at the 5’ or 3’ end of your minigene. In case minigenes will also be used for protein analysis, this will enable a convenient visualization of protein expression. Check for the correct reading frame and the presence of a start codon and stop codon at appropriate positions by sequencing prior to proceeding with the next steps.
      2. This vector is required for initial in vitro testing of the minigene expression and validation of mRNA splicing. Do not use rAAV vectors for cloning at this stage, as the site-directed mutagenesis (see the following section) will be prevented by the secondary structure of the rAAV vector ITRs.

  4. Site-directed mutagenesis to introduce point mutations of interest into minigenes
    D1. Design of mutagenic oligonucleotide primers
    1. Use the QuikChange primer design program and select the QuikChange kit that you require (e.g., QuikChange Lightning).
    2. Paste the DNA sequence of your gene of interest in the box, then click on ‘Upload now’.
    3. To generate a point mutation select the nucleotide to be changed by ticking the corresponding box. Subsequently, choose the substituting nucleotide in the box of ‘Site 1’.
    4. Click on ‘Design Primers’.
    5. Obtain the sequence of the forward and reverse primer and additional information (e.g., length, Tm).
    6. Order primers from commercial providers (e.g., Eurofins Genomics).
      Notes:
      1. The desired mutation should lie within the middle of both primers with flanking 10 to 15 bp of correct sequence on each side.
      2. Their length should be between 25 and 45 bp.
      3. Tm ≥ 78 °C (verify by Tm calculator, e.g., NEB)

    D2. Mutant strand synthesis reaction
    1. Prepare the sample reaction as follows:
      5 µl
      10x QuikChange Lightning Buffer*
      X µl
      10-100 ng of dsDNA template
      X µl
      125 ng of forward primer
      X µl
      125 ng of reverse primer
      1 µl
      dNTP mix*
      1.5 µl
      QuickSolution reagent*
      1 µl
      QuickChange Lightning Enzyme*
      Add to 50 µl
      Double distilled H2O
      Notes:
      1. *Provided in the kit (Materials and Reagents C2).
      2. We recommend 10-30 ng of dsDNA template.
    2. Mix all reagents in a PCR tube by pipetting up and down.
    3. For the PCR use following cycling parameters:

      Note: For optimal results the annealing temperature can be adapted as necessary.
    4. Check for successful amplification by loading 3-5 µl of the PCR product on an agarose gel.

    D3. DpnI digestion of template
    1. Add 2 µl of the provided DpnI restriction enzyme to the amplification reaction and mix the solution by pipetting up and down.
    2. Incubate the mixture at 37 °C for 1-2 h to digest the non-mutated, methylated template DNA.

    D4. Transformation
    1. Thaw an aliquot (45 µl) of the provided XL10-Gold ultracompetent cells (or an alternative strain of competent cells) on ice.
    2. Transfer 2-4 µl of the DpnI-treated DNA to the cells and swirl gently to mix the components.
    3. Incubate the transformation reaction on ice for 30 min.
    4. Heat-pulse the tube at 42 °C for 30 sec.
    5. Incubate the tube on ice for 2 min.
    6. Plate the whole transformation reaction on an agar plate containing the appropriate antibiotic for the plasmid vector.
    7. Incubate agar plate at 37 °C for >16 h.

    D5. Picking colonies, plasmid isolation, digestion
    1. Pick 24 well-separated colonies from the agar plate and inoculate culture tubes containing 5 ml LB medium and the appropriate concentration of antibiotic. Shake at 250 rpm and 37 °C for > 7 h.
    2. Isolate plasmid DNA using alkaline lysis or a plasmid miniprep kit according to the manufacturer’s instructions.
    3. Digest the isolated DNA with appropriate restriction enzymes to verify the presence of your minigene in the plasmid.
      Note: Check whether site-directed mutagenesis creates new or deletes existing restriction sites. If this is the case, the digestion with appropriate restriction enzymes directly allows determining the success of the QuikChange PCR.
    4. Sequence to confirm the correct plasmids prior to use.

  5. Subcloning of minigenes into rAAV cis vector plasmid
    Use appropriate restriction sites to clone your mutant minigenes into a rAAV vector using standard cloning techniques.
    Note: Check for ITR integrity prior to rAAV production. The AAV ITRs contain SmaI (or XmaI) sites, which can be used as diagnostics for ITR integrity.

Notes

  1. Not all genes are suitable for an in vivo splice analysis using rAAV-mediated gene expression. If the size of the expression cassette containing a promoter, the minigene and a poly-A signal exceeds 4.7 kb, lentiviral-derived vector systems might be used.
  2. Instead of PCR-based cloning, the minigenes can also be ordered via gene synthesis from commercial providers.
  3. In the case of using minigenes for mRNA splicing only, the exon to be analyzed should be flanked by at least two additional native exons including the respective full-length or shortened intronic sequences. In contrast to the middle exon, the remaining two exons do not necessarily need to be flanked with the corresponding intronic sequence on both sides.
  4. As deep intronic regions might also contain splice regulatory elements, shortening of the native introns should be done only if necessary.
  5. In case no PCR products are obtained for some overlap PCRs, consider varying the length of the overlap region. Alternatively, change the melting temperature and/or the order of the template PCR amplicons used for initial overlap PCRs in Step C1.2 (cf. Figure 2).

Recipes

  1. 1x TBE buffer

  2. LB-medium/LB agar plates

    Mix all reagents. If making agar plates, also add 15 g agar to the solution. Autoclave the solution at 121 °C for 10 min. If making selective media or agar plates, add the desired antibiotic in the appropriate concentration once the medium has cooled down to 50 °C. For agar plates, pour the medium into Petri dishes

Acknowledgments

We thank Berit Noack for the valuable technical support. This work was funded by the Deutsche Forschungsgemeinschaft, grant number BE 4830/1-1. The authors declare that they have no competing interests.

References

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  2. Becirovic, E., Bohm, S., Nguyen, O. N., Riedmayr, L. M., Koch, M. A., Schulze, E., Kohl, S., Borsch, O., Santos-Ferreira, T., Ader, M., Michalakis, S. and Biel, M. (2016b). In vivo analysis of disease-associated point mutations unveils profound differences in mRNA splicing of peripherin-2 in rod and cone photoreceptors. PLoS Genet 12(1): e1005811.
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  16. Zincarelli, C., Soltys, S., Rengo, G. and Rabinowitz, J. E. (2008). Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection. Mol Ther 16(6): 1073-1080.

简介

影响mRNA剪接的疾病相关突变称为剪接突变。大多数剪接突变位于确定典型供体和受体剪接位点的外显子 - 内含子边界上。然而,编码区中的突变(外显子突变)也可影响mRNA剪接。准确了解剪接突变的疾病机制对于开发最佳治疗策略至关重要。鉴于迄今为止鉴定的大量疾病相关突变,尚未满足对系统分析致病突变对mRNA剪接的影响的方法的需求。由于不同细胞类型之间的拼接可能不同,如果可能的话,拼接突变需要在天然条件下进行测试。一种常用的分析mRNA剪接的工具是携带外显子和内含子序列的小基因的构建。在这里,我们描述了设计和克隆到重组腺相关病毒(rAAV)载体中用于基因递送和在本地环境中调查mRNA剪接的方案。该协议是为了基于小基因的视网膜细胞中mRNA剪接分析而开发的,但是原则上它适用于任何可以用rAAV载体转导的细胞类型。

【背景】预计大部分疾病相关突变(至少15%)会导致异常的mRNA剪接(Cartegni等,2002; Singh和Cooper,2012; Sterne-Weiler和Sanford,2014年)。 '经典'剪接突变是影响定义5'和3'剪接位点(分别为供体和受体剪接位点)的规范序列的突变。然而,剪接突变也可能发生在其他非编码区和编码区(Wang和Cooper,2007; Scotti和Swanson,2016)。越来越多的证据表明,编码区(外显子突变)中剪接突变的频率被低估(Julien等人,2016; Soukarieh等人,2016)。外显子剪接突变(即突变,点突变,插入或缺失)可诱导外显子跳跃,内含子保留或导致产生新的供体或受体剪接位点。根据基因和外显子的组成,这些机制可能对从haploinsufficiency到获得功能的蛋白质水平有不同的影响。尽管如此,支持致病突变的分子机制的确切知识对于开发最佳治疗方法至关重要。

mRNA剪接以高度细胞类型特异性方式发生,强调需要分析主要受突变影响的组织中潜在剪接突变的影响(Wang等人,2008)。因此,在最佳情况下,应该在天然基因和天然组织中分析mRNA剪接。然而,这个选项的要求很高,原因如下:
1)它可能需要精心制作的基因修饰人类细胞系。这妨碍了对单个基因的剪接突变的更系统的分析。
2)许多天然细胞类型是高度专业化的,它们的培养对应物(如果有的话)不反映天然细胞的各种形态和分子标志,包括剪接机器的组成和活性。
3)在给定组织中产生表达各自剪接突变的人源化动物模型不仅在技术上具有挑战性,而且耗时且昂贵。因此,这种方法似乎也不适合系统检测给定基因的剪接突变。
4)通常,天然基因太大而无法克隆到经典表达载体中。

避开这些障碍中的一些的一个替代方案是使用人类小基因来设计用于在合适的动物模型中表达(例如,小鼠)。我们在最近的研究中评估了这种方法,其解决了mRNA剪接中不同基因(例如PRPH2)中疾病相关突变的影响(Becirovic等人, 2016年; Nguyen等人,2016年; Khan 等人,2017年; Petersen-Jones等人,2017年) 。对于小基因的稳定和特异性异位表达,我们利用了rAAV载体。这些载体能够在体内转导各种不同的细胞类型(Zincarelli等人,2008; Lisowski等人,2014) 。此外,rAAV载体的设计,克隆,生产和纯化可在几周内完成,且不需要复杂的技术设备(Becirovic等,2016a)。

包括PRPH2的大多数天然基因超过了AAV的有限包装容量(约4.7kb)(Wu等人,2010)。因此,我们设计了缺少大内含子部分的小基因,这通常不包含正确的mRNA剪接所需的信息。对于不含大外显子数目或大小的基因,内含子切片的缩短也允许将整个蛋白质编码区引入基于rAAV载体的小基因。

该策略(参见图1)被开发和评估以分析已知疾病相关突变对光接受者特异性基因中mRNA剪接和蛋白质表达的影响,但原则上也应该可转移到其他细胞类型。

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图1.构建和克隆小基因的工作流程示意图

关键字:微小基因, mRNA剪接, 克隆, 腺相关病毒, 分析突变

材料和试剂

  1. 分离人基因组DNA克隆小基因

    1. 1.5 ml Eppendorf管(SARSTEDT,目录号:72.690.001)
    2. 手术刀(斯旺莫顿,目录号:0510)
    3. Gentra Puregene口腔细胞试剂盒(QIAGEN,目录号:158845)
    4. 2-丙醇(100%,ACS,ISO,Reag。Ph.Eur。等级)
    5. 乙醇(70%,ACS,ISO,Reag。Ph.Eur。等级)

  2. 重叠延伸PCR用于构建和克隆小基因
    1. PCR管(品牌,目录号:781320和781334)
    2. 选择的表达载体(例如,,pcDNA3.1载体(+),Thermo Fisher Scientific,Invitrogen TM,目录号:V79020)
    3. 双蒸H 2 O(ddH 2 O)
    4. Herculase II融合DNA聚合酶(Agilent Technologies,目录号:600675)
    5. dNTPs,10mM(Thermo Fisher Scientific,Thermo Scientific TM,目录号:R0192)
    6. DNA加载染料(Thermo Fisher Scientific,Thermo Scientific TM,目录号:R0611)
    7. GeneRuler 1 kb Plus DNA Ladder(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:SM1331)
    8. EDTA(VWR,目录号:20302.293)
    9. 三(羟甲基)氨基甲烷(VWR,目录号:103156X)
    10. 硼酸(VWR,目录号:20185.360)
    11. QIAquick凝胶提取试剂盒(QIAGEN,目录号:28704)
    12. 1x TBE缓冲液(见食谱)

  3. 定点诱变以将感兴趣的点突变引入小基因
    1. PCR管(品牌,目录号:781320和781334)
    2. QuikChange Lightning定点诱变试剂盒(Agilent Technologies,产品目录号:210518)
    3. 双蒸H 2 O(ddH 2 O)
    4. 蛋白胨(Applichem,目录号:403898.1210)
    5. 酵母提取物(Applichem,目录号:A1552)
    6. NaCl(VWR,目录号:27810.364)
    7. D - (+) - 葡萄糖(Sigma-Aldrich,目录号:49159)
    8. 琼脂(Sigma-Aldrich,目录号:A5054)
    9. 适用于质粒载体的抗生素(例如,氨苄青霉素,卡尔罗斯,目录号:K029.2)
    10. 含有用于质粒载体的合适抗生素的LB琼脂平板(参见食谱)
    11. LB培养基(见食谱)

  4. 将小基因亚克隆到rAAV载体中
    1. 适当的rAAV载体质粒(如emA,pAAV-MCS2,Addgene,目录号:46954)

设备

  1. 移液器(如,Eppendorf)
  2. Vortexer(Heidolph Instruments,型号:Reax top)
  3. 培养箱(Thermo Fisher Scientific,Thermo Scientific TM,型号:Heraeus B12 Function Line,目录号:50042307)
  4. 微量离心机(Eppendorf,型号:Centrifuge MiniSpin ,目录号:5452000018)
  5. 两个水浴(Haake,目录号:003-2859)
  6. 热循环仪(Thermo Fisher Scientific,型号:ProFlex TM,目录号:4483636)
  7. NanoDrop TM(Thermo Fisher Scientific,Thermo Scientific TM,目录号:ND-2000C)

软件

  1. 人类拼接发现软件( eg , http://www.umd.be/HSF3/
  2. 计算器软件(例如,NEB T m 计算器)

程序

  1. 电脑模拟拼接分析
    在很多情况下,初步预测单个突变的mRNA剪接可能是有帮助的。为了预测对感兴趣突变的剪接的潜在影响,可以使用人类剪接发现软件( , http://www.umd.be/HSF3/ )。
    1. 转到 http://www.umd.be/HSF3/ 并选择分析类型。我们建议使用“分析序列”选项。
    2. 选择一个序列。我们建议使用“粘贴自己的序列”选项。将感兴趣的序列的野生型版本粘贴到提供的框中,然后点击“继续分析”。
      注意:您的序列应在两侧各自的点突变的侧翼至少包含40 bp。
    3. 点击'执行快速突变',输入您感兴趣的突变相对于之前输入的序列的位置并选择新的核苷酸。然后点击“继续快速突变分析”。
    4. 人类接头查找器网络服务器输出:
      1. 点击'原始数据表'来详细分析输入的点突变的影响。
      2. 为您提供了五个不同的数据表,称为“潜在剪接位点”,“潜在分支点”,“增强子图案”,“消声器图案”和“其他剪接图案”。它们中的每一个都显示由点突变引起的百分比变化。新形成的剪接位点或图案用绿色表示,现有位置或图案的强度变化用浅黄色表示。
    5. 对所有感兴趣的点突变重复进行分析,并选择预测影响剪接的基因以进一步克隆。

  2. 分离人基因组DNA克隆小基因

    1. 将一个水浴预热至65°C,另一个预热至37°C
    2. 为了收集颊细胞,用Buccal Collection Brush(在试剂盒,材料和试剂A3中提供)刮擦口腔内部10次。
      注意:为获得最佳效果,在进食或饮水后至少等待1小时以收集颊细胞。
    3. 将300μl细胞裂解溶液(在试剂盒中提供,材料和试剂A3)分配到1.5ml Eppendorf管中。
      使用无菌手术刀从手柄上取下收集刷,并将分离的头放入管中。
    4. 在65°C孵育60分钟以完成细胞裂解。
    5. 从细胞溶解液中取出收集刷头,在管侧刮取尽可能多的液体。
    6. 加入1.5μlRNase A Solution(试剂盒中提供),并将试管翻转25次。
    7. 37°C孵育30分钟。在冰上孵育1分钟以冷却样品。
    8. 加入100μl蛋白质沉淀溶液(在试剂盒中提供,材料和试剂A3),高速涡流剧烈搅拌20秒。

    9. 在冰上孵育5分钟
    10. 在13,000-16,000×g的条件下离心3分钟沉淀沉淀的蛋白质。
    11. 吸取300μl异丙醇和0.5μl糖原溶液(在试剂盒中提供,材料和试剂A3)到一个干净的1.5ml Eppendorf管中,并且通过倾倒或小心移液加入上一步的上清液。确保不要转移蛋白质颗粒。

    12. 轻轻翻转试管50次或以20rpm旋转2.5分钟混合。

    13. 在RT下离心5分钟,13,000-16,000×g 。
    14. 小心丢弃上清液,并将管倒置在一块干净的吸水纸上,注意将颗粒留在管中。
    15. 加入300μl的70%乙醇,反转几次,洗涤DNA沉淀。
    16. 在RT下离心1分钟,13,000-16,000×g 。
    17. 小心丢弃上清液。将管子放在一张干净的吸水纸上,注意颗粒留在管中。允许风干5分钟。
    18. 加入100μlDNA水合溶液(在试剂盒中提供,材料和试剂A3),并以中等速度涡旋5秒以混合。

    19. 在65°C孵育1小时以溶解DNA。
    20. 在室温下温和振荡孵育过夜。确保管帽紧闭以避免泄漏。然后将样品短暂离心并转移到储存管中。

  3. 重叠延伸PCR用于构建和克隆小基因
    的 C1。重叠引物的方法描述和设计
    为了使基于小基因的mRNA拼接突变分析能够缩短大内含子。在我们最近的研究中,我们验证了约。 200bp的外显子区域位于外显子侧翼足以进行正确的mRNA剪接(Becirovic等人,2016b; Nguyen等人,2016)。
    为了通过标准克隆程序产生合适的小基因,可以使用重叠延伸PCR。为此,作为第一步,必须通过标准PCR扩增各自的外显子,包括≥200bp的侧翼内含子。用于此PCRs的引物必须包含约定义的突出端。 20 bp。这些突出端与待在第二重叠延伸PCR中连接的相应PCR片段互补。这些产生了用于扩增两个片段的〜40bp重叠区。我们将这一策略应用于创建包含三个本地外显子的小基因,其中包括两个缩短的侧翼内含子(图2)。然而,这种方法也适用于具有随机数目的外显子和缩短的内含子的小基因的产生。
    1. 设计PCR引物以扩增一个外显子加上≥200bp的侧翼内含子区域。使用T计算器软件(例如,NEB T计算器)检查熔化温度(Tm / 2) 。
    2. 为了创建重叠区域,引物必须包含约。与邻接的PCR扩增子互补的20bp长的区域(见图2)。
      例如:当设计引物扩增第二外显子加侧翼内含子区域(扩增子#2)时,将含有外显子1的扩增子(扩增子#1)中最后20 bp的序列添加到正向引物的5'末端。接下来,将含有外显子3的第一个20bp的扩增子(扩增子#3)的反向序列添加到反向引物的5'末端。这些重叠序列对于后续重叠延伸PCR将是必需的。


      图2.重叠延伸PCR的示意图,用于创建包含三个外显子和两个缩短的侧翼内含子的小基因。 单个扩增子以不同的颜色显示。限制网站显示为蓝色。引物根据它们的模板和匹配的重叠被描述为双色。来自步骤1的两个PCR扩增子用作重叠PCR1的模板,并且可以以随机方式(例如,PCR1 + PCR2的扩增子)组合在一起。最后的重叠PCR 2可以用剩余的PCR片段(在本例中为PCR 3的扩增子)进行。对于所有重叠PCR,只应使用第二个PCR扩增子的第一个和第三个引物的5'引物。

    3. 对于第一个小基因外显子的扩增:根据您的表达载体的限制性位点(例如, Hind )添加适当的限制性位点到正向引物的5' III在pcDNA3.1中)。考虑在限制性酶切位点的5'末端添加一个额外的序列(最多4 bp),以便通过限制酶进行更有效的切割。
      对于最后一个小基因外显子的扩增:根据您的表达载体的限制性位点(例如, Not I )。考虑在限制性酶切位点的5'末端添加高达4 bp的限制性内切酶进行更有效的切割。
    4. 从商业供应商(例如Eurofins Genomics)订购PCR引物(大约0.05μmol,至少HPSF纯化的)。

    的 C2。 PCR扩增全长外显子和缩短内含子
    1. 为了扩增单个外显子和侧翼内含子区域,按如下方法制备样品反应:
      X µl
      Genomic DNA (100-300 ng)
      5 µl
      5x Herculase II Fusion buffer*
      1 µl
      dNTPs (10 mM)
      1 µl
      Forward primer (10 µM)
      1 µl
      Reverse primer (10 µM)
      0.25 µl
      Herculase II Fusion DNA Polymerase*
      Add to 25 µl
      Double distilled H2O
      *注意:在试剂盒中提供(材料和试剂B4)。

    2. 上下移液,将所有试剂混合在PCR管中
    3. 对于PCR,使用以下循环参数:

      注:为获得最佳效果,退火温度可根据需要进行调整。
    4. 扩增后,加入5μl6x DNA Loading Dye到您的PCR反应中,加入0.7%琼脂糖凝胶以及您选择的DNA标准品(例如GeneRuler 1 kb Plus DNA Ladder)并运行它在1x TBE缓冲液中。检查正确的放大。
    5. 如果扩增成功,即如果你看到一个正确大小的条带,用一把手术刀切除它,然后将凝胶片放入Eppendorf管中。
      根据制造商的说明使用凝胶提取试剂盒纯化DNA
    6. 使用NanoDrop测量DNA浓度。
    7. 对所有外显子重复步骤C2。

    的 C3。重叠延伸PCR
    1. 要加入包含外显子1和2的前两个扩增子,请按以下步骤准备样品反应:
      X µl
      Amplicon #1 (100-150 ng)
      X µl
      Amplicon #2 (use molar ratio 1:1)
      5 µl
      5x Herculase II Fusion buffer
      1 µl
      dNTPs (10 mM)
      1 µl
      Forward primer of Amplicon #1 (10 µM)
      1 µl
      Reverse primer of Amplicon #2 (10 µM)
      0.25 µl
      Herculase II Fusion DNA Polymerase
      Add to 25 µl
      Double distilled H2O

    2. 上下移液,将所有试剂混合在PCR管中
    3. 对于PCR,使用以下循环参数:

      注:为获得最佳效果,可根据需要调整退火温度。
    4. 将所有的PCR反应加载到琼脂糖凝胶上并检查扩增是否正确。
    5. 如果扩增成功,即如果您看到正确大小的条带,请根据制造商的说明使用凝胶提取试剂盒切下条带并纯化DNA。
    6. 使用NanoDrop测量DNA浓度。
    7. 重复 重叠延伸PCR 以连接新的扩增子和随后的扩增子。为此,使用两种扩增子的纯化DNA作为模板;并且扩增子#1的正向引物和扩增子#3的反向引物作为重叠延伸PCR引物。
    8. 根据要连接在一起的扩增子的数量,可以重复步骤1-7。
    9. 使用标准克隆技术,使用选定的限制性位点将您的小基因克隆到相应的表达载体中。
    10. 通过测序(例如,Eurofins Genomics)检查小基因的正确序列。
      注意:
      1. 考虑在小基因的5'或3'末端使用携带报道基因(例如GFP)的表达载体。如果小基因也将用于蛋白质分析,这将使得蛋白质表达的方便可视化成为可能。

        检查正确的阅读框,并在进行下一步之前,通过测序在适当的位置存在起始密码子和终止密码子。
      2. 该载体对于小基因表达的初始体外测试和mRNA剪接的验证是必需的。在此阶段不要使用rAAV载体进行克隆,因为rAAV载体ITRs的二级结构会阻止定点诱变(参见下节)。

  4. 定点诱变将感兴趣的点突变导入小基因
    的 D1。诱变寡核苷酸引物的设计
    1. 使用 QuikChange引物设计程序并选择您需要的QuikChange试剂盒( eg ,QuikChange Lightning)。
    2. 将您感兴趣的基因的DNA序列粘贴到框中,然后点击“立即上传”。
    3. 要生成点突变,请通过勾选相应的框来选择要更改的核苷酸。随后,选择“Site 1”框中的替代核苷酸。
    4. 点击'设计入门'。
    5. 获得正向和反向引物的顺序以及附加信息(例如,长度,T m )。
    6. 从商业供应商(例如,Eurofins Genomics)订购引物。
      注意:
      1. 期望的突变应位于两侧引物的中间,两侧各有10至15 bp的正确序列。
      2. 它们的长度应该在25到45 bp之间。
      3. m ≥78°C(由Tm计算器验证,例如NEB)

    的 D2。突变链合成反应
    1. 准备样品反应如下:
      5 µl
      10x QuikChange Lightning Buffer*
      X µl
      10-100 ng of dsDNA template
      X µl
      125 ng of forward primer
      X µl
      125 ng of reverse primer
      1 µl
      dNTP mix*
      1.5 µl
      QuickSolution reagent*
      1 µl
      QuickChange Lightning Enzyme*
      Add to 50 µl
      Double distilled H2O
      注意:
      1. *在试剂盒中提供(材料和试剂C2)。
      2. 我们推荐10-30 ng双链DNA模板。

    2. 上下移液,将所有试剂混合在PCR管中
    3. 对于PCR使用以下循环参数:

      注:为获得最佳效果,可根据需要调整退火温度。
    4. 通过在琼脂糖凝胶上加载3-5μl的PCR产物检查扩增是否成功。

    的 D3。 DpnI消化模板
    1. 向扩增反应液中加入2μl所提供的限制性内切酶,并通过上下移液来混合溶液。
    2. 将混合物在37℃孵育1-2小时以消化未突变的甲基化模板DNA。

    的 D4。转型
    1. 在冰上解冻提供的XL10-Gold超感受态细胞(或另一种感受态细胞株)的等分试样(45μl)。
    2. 将2-4μl的Dpn I处理的DNA转移到细胞中并轻轻旋转以混合组分。

    3. 在冰上培养转化反应30分钟

    4. 在42°C加热脉冲管30秒。
    5. 在冰上孵育2分钟。
    6. 将整个转化反应平铺在含有用于质粒载体的合适抗生素的琼脂平板上。
    7. 在37℃孵育琼脂平板> 16小时。

    的 D5。挑选菌落,质粒分离,消化
    1. 从琼脂平板挑取24个很好分离的菌落并接种含有5ml LB培养基和适当浓度的抗生素的培养管。以250rpm和37℃摇动> 7小时。
    2. 根据制造商的说明书,使用碱裂解或质粒小量制备试剂盒分离质粒DNA。
    3. 用适当的限制性内切酶消化分离的DNA,以验证质粒中小基因的存在。
      注:检查定点突变是否会产生新的或删除现有的限制性位点。如果是这种情况,用适当的限制酶直接消化可以确定QuikChange PCR的成功。
    4. 在使用前确认正确质粒的序列。

  5. 将小基因亚克隆到rAAV cis载体质粒中 使用适当的限制性位点,使用标准克隆技术将您的突变小基因克隆到rAAV载体中。
    注:在rAAV生产之前检查ITR完整性。 AAV ITR包含SmaI(或XmaI)位点,可用作ITR完整性的诊断。

笔记

  1. 不是所有的基因都适合用rAAV介导的基因表达进行体内剪接分析。如果含有启动子,小基因和poly-A信号的表达盒的大小超过4.7kb,则可以使用慢病毒来源的载体系统。
  2. 代替基于PCR的克隆,小基因也可以通过基因合成从商业供应商订购。
  3. 在仅使用小基因进行mRNA剪接的情况下,待分析的外显子应侧接至少两个额外的天然外显子,包括各自的全长或缩短的内含子序列。与中间外显子形成对比的是,其余两个外显子不一定需要两侧都有相应的内含子序列。
  4. 由于深部内含子区域也可能含有剪接调控元件,因此只有在必要时才能缩短天然内含子。
  5. 如果某些重叠PCR没有获得PCR产物,请考虑改变重叠区域的长度。或者,改变步骤C1.2中用于初始重叠PCR的模板PCR扩增子的熔解温度和/或顺序(参见图2)。

食谱

  1. 1x TBE缓冲液

  2. LB培养基/ LB琼脂平板

    混合所有试剂。如果制作琼脂平板,也可以在溶液中加入15克琼脂。将溶液在121℃高压灭菌10分钟。如果制作选择性培养基或琼脂平板,一旦培养基冷却至50°C后,加入适当浓度的所需抗生素。对于琼脂平板,将培养基倒入培养皿中

致谢

我们感谢Berit Noack宝贵的技术支持。这项工作由Deutsche Forschungsgemeinschaft资助,授权号为BE 4830 / 1-1。作者声明他们没有竞争利益。

参考

  1. Becirovic,E.,Bohm,S.,Nguyen,ON,Riedmayr,LM,Hammelmann,V.,Schon,C.,Butz,ES,Wahl-Schott,C.,Biel,M.and Michalakis,S.(2016a )。 用于基于FRET的光感受器外节段蛋白质 - 蛋白质相互作用分析的AAV载体。
    前面的Neurosci 10:356.
  2. Becirovic,E.,Bohm,S.,Nguyen,ON,Riedmayr,LM,Koch,MA,Schulze,E.,Kohl,S.,Borsch,O.,Santos-Ferreira,T.,Ader,M.,Michalakis ,S.和Biel,M。(2016b)。 体内分析疾病相关的点突变揭示了mRNA中的深刻差异在杆状和锥状感光器中剪接周边蛋白-2。 PLoS Genet 12(1):e1005811。
  3. Cartegni,L.,Chew,S.L。和Krainer,A.R。(2002)。 倾听沉默和理解废话:影响剪接的外显子突变 Nat Rev Genet 3(4):285-298。
  4. Julien,P.,Minana,B.,Baeza-Centurion,P.,Valcarcel,J.和Lehner,B。(2016)。 人类外显子选择性剪接的完整局部基因型 - 表型格局 Nat Commun 7:11558。
  5. Khan,AO,Becirovic,E.,Betz,C.,Neuhaus,C.,Altmuller,J.,Maria Riedmayr,L.,Motameny,S.,Nurnberg,G.,Nurnberg,P.和Bolz,HJ(2017 )。 深部内含子CLRN1(USH3A)的创始人突变引发异常外显子,并成为阿拉伯人严重Usher综合征的基础半岛。 Sci Rep 7(1):1411.
  6. Lisowski,L.,Dane,A.P.,Chu,K.,Zhang,Y.,Cunningham,S.C.,Wilson,E.M.,Nygaard,S.,Grompe,M.,Alexander,I.E。和Kay,M.A。(2014)。 选择和评估异种移植肝脏模型中临床相关的AAV变异体 自然 506(7488):382-386。
  7. Nguyen,O.N。,Bohm,S.,Giessl,A.,Butz,E.S。,Wolfrum,U.,Brandstatter,J.H。,Wahl-Schott,C.,Biel,M.and Becirovic,E.(2016)。 Peripherin-2与锥形光感受器外层锥形视蛋白有差别的相互作用 Hum Mol Genet 25(12):2367-2377。
  8. Petersen-Jones,SM,Occelli,LM,Winkler,PA,Lee,W.,Sparrow,JR,Tsukikawa,M.,Boye,SL,Chiodo,V.,Capasso,JE,Becirovic,E.,Schön,C. ,Seeliger,MW,Levin,AV,Michalakis,S.,Hauswirth,WW和Tsang,SH(2017)。 CNGβ1缺陷型视网膜色素变性支持基因增强方法的患者和动物模型 JCI 128(1)。
  9. Scotti,M.M。和Swanson,M.S。(2016)。 RNA错误拼接疾病 Nat Rev Genet 17(1):19-32。
  10. Singh,R.K。和Cooper,T.A。(2012)。 前体mRNA在疾病和治疗中的剪接。 趋势Mol Med < (18):472-482。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Riedmayr, L. M., Böhm, S., Michalakis, S. and Becirovic, E. (2018). Construction and Cloning of Minigenes for in vivo Analysis of Potential Splice Mutations. Bio-protocol 8(5): e2760. DOI: 10.21769/BioProtoc.2760.
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