2 users have reported that they have successfully carried out the experiment using this protocol.
Assaying the Effects of Splice Site Variants by Exon Trapping in a Mammalian Cell Line

Jia Li
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The Journal of Clinical Investigation
Jul 2016



There are several in silico programs that endeavor to predict the functional impact of an individual’s sequence variation at splice donor/acceptor sites, but experimental confirmation is problematic without a source of RNA from the individual that carries the variant. With the aid of an exon trapping vector, such as pSPL3, an investigator can test whether a splice site sequence change leads to altered RNA splicing, through expression of reference and variant mini-genes in mammalian cells and analysis of the resultant RNA products.

Keywords: Splicing (剪接), Mutation (突变), Exon (外显子), Trapping (捕获), Expression (表达), Transcript (转录), Variant (变体), Splice (剪接物)


We wished to experimentally test the functional impact of two splice donor site variants, c.760+2T>C and c.3300+2delT, identified in the TEK gene (Souma et al., 2016). As is often the case, samples of cells or mRNA were not available from the individuals carrying these sequence variants, so we utilized the exon trapping method to serve as a functional test. DNA samples were available from patients for PCR amplification of the genomic regions of interest. If patient gDNA samples are unavailable, sequence variants can also be incorporated into wild-type sequence by methods such as PCR-based site-directed mutagenesis.

The exon trapping approach was originally developed to identify unknown exons within long stretches of genomic DNA (Duyk et al., 1990). The pSPL3 exon trapping vector was created to increase the efficiency and reliability of exon identification, and also allowed larger genomic fragments to be screened (Church et al., 1994; Nisson et al., 1994). The pSPL3 vector contains a small artificial gene composed of an SV40 promoter, an exon-intron-exon sequence with functional splice donor and acceptor sites, and a late polyadenylation signal. Within the single intron a multiple cloning site is located, into which a genomic fragment of interest is inserted to create a mini-gene expression construct.

In our example, patient and control genomic DNA fragments from the TEK gene were PCR amplified and cloned between pSPL3 vector exons V1 and V2 using XhoI and BamHI restriction sites. COS-7 cells were then transfected with the mini-gene constructs and the resulting RNA content purified. mRNA transcripts were then reverse transcribed into cDNA. Using vector exon-specific primers, cDNAs produced from the mini-gene constructs were specifically PCR amplified and Sanger sequenced. For the first splice site variant, c.760+2T>C within the 5’ splice site of exon 5, a 1,457 bp genomic fragment of the TEK gene encompassing all of intron 4, exon 5, intron 5, exon 6 and intron 6 was inserted into the construct (Figure 1A). RT-PCR and Sanger sequencing of the mini-gene expressed transcripts showed that the mutation destroyed the splice donor site, which resulted in partial intron 5 inclusion before a cryptic splice site was utilized (Figure 1C). This splicing error is predicted to result in a translational frameshift and premature termination signal, which would likely lead to transcript elimination via the nonsense-mediated decay pathway. For the second splice site variant, c.3300+2delT within the 5’ splice site of exon 22, an 831 bp genomic fragment of the TEK gene encompassing all of intron 21, exon 22 and intron 22 was inserted into the construct (Figure 1B). RT-PCR and Sanger sequencing of the mini-gene expressed transcripts revealed that the splice donor mutation led to skipping of exon 22, which is also predicted to result in a translational frameshift and premature termination signal in the genomic context of the patient (Figure 1C).

Figure 1. Exon trapping assay. Vector exons V1 and V2, are depicted as black boxes and TEK exons 5, 6, and 22 are shown in gray. Vector exon-specific primers are indicated by half-arrows in (A) and (B). Wild-type (WT) and mutant (M) splicing products, with included exon sizes in base pairs, are indicated by dashed lines above and below the construct, respectively. The locations of the splice site mutations are shown as an asterisk (*). A. Wild-type (WT-5) and mutant (M-5) genomic fragments containing TEK exons 5 and 6 were used to model the c.760+2T>C mutation. B. Wild-type (WT-22) and mutant (M-22) genomic fragments containing TEK exon 22 were used to model the c.3300+2delT mutation. C. Gel electrophoresis of RT-PCR products from transfected COS-7 cells. ‘Empty Vector’, cells transfected with vector containing no gDNA insert; ‘TF –ve’ (transfection negative), cells transfected with QIAGEN buffer EB only; ‘PCR –ve’ (PCR negative), PCR contamination control substituting water for cDNA template. Wild-type and mutant transcript content, determined by Sanger sequencing, is depicted to the right of the gel image. The additional 21 bp of intron 5 sequence identified within the M5 transcript is shown incorporating a premature termination codon between exons 5 and 6.

Materials and Reagents

  1. PCR
    1. 0.2 ml PCR tubes with caps (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM12225 )
    2. 1.5 ml conical base screw cap tube (USA Scientific, catalog number: 1415-8399 )
    3. Human genomic DNA samples with and without splice site variant (20 ng/μl)
    4. TE buffer, 10 mM Tris 1 mM EDTA pH 8.0 (Sigma-Aldrich, catalog number: 93283 )
    5. Custom-synthesized oligonucleotide primers incorporating restriction endonuclease sites to the 5’ end (Integrated DNA Technologies; https://www.idtdna.com/site/order/oligoentry):

        Note: Genome-specific sequences are underlined, restriction sites are shown in BOLD and an extra 8 bp added to the 5’ end are shown in lowercase. 5’-CTCGAG-3’ is the recognition sequence for XhoI and 5’-GGATCC-3’ is the recognition sequence for BamHI. ‘-F’ and ‘-R’ refers to the forwards and reverse primers in a pair, respectively. Resuspend primers and make 10 μM stocks with TE buffer.

    6. Phusion Hot Start II High-Fidelity DNA polymerase (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: F549S )
    7. Molecular grade water, nuclease-free (Dot Scientific, catalog number: DS248700 )
    8. dNTP Set (QIAGEN, catalog number: 201913 )
    9. 2 mM dNTPs (see Recipes)

  2. Gel electrophoresis
    1. 1 M Trizma hydrochloride solution, Tris-HCl, pH 7.8 (Sigma-Aldrich, catalog number: T2569 )
    2. 3 M sodium acetate buffer solution, pH 5.2 (Sigma-Aldrich, catalog number: S7899 )
    3. 0.5 M EDTA, pH 8.0 (Santa Cruz Biotechnology, catalog number: sc-203932 )
    4. Agarose (IBI Scientific, catalog number: IB70042 )
    5. SYBR safe DNA gel stain (Thermo Fisher Scientific, InvitrogenTM, catalog number: S33102 )
    6. Ficoll-400 (Dot Scientific, catalog number: DSF10400-25 )
    7. Bromophenol blue, sodium salt (MP Biomedicals, catalog number: 02152506 )
    8. Orange G (Sigma-Aldrich, catalog number: O3756 )
    9. HyperLadder 1 kb (Bioline, catalog number: BIO-33053 )
    10. 1x TAE buffer (see Recipes)
    11. 1% or 1.5% agarose gel (see Recipes)
    12. 10x gel loading dye (see Recipes)

  3. Cloning
    1. 1.5 ml Eppendorf tubes (VWR, catalog number: 20170-022 )
    2. Petri dishes, 100 x 15 mm (VWR, catalog number: 25384-302 )
    3. Whatman GD/X syringe filters, 0.2 μm pore size (Whatman, catalog number: 6901-2502 )
    4. BD 60 ml syringes, Luer-Lok Tip (BD, catalog number: 309653 )
    5. Pasteur glass pipettes, 230 mm (for spreading bacteria on plates) (WHEATON, catalog number: 357335 )
    6. 2 ml cryovial, self-standing (Simport, catalog number: T310-2A )
    7. 50 ml polypropylene conical tube, Falcon (Corning, Falcon®, catalog number: 352070 )
    8. 0.5 ml PCR tubes (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM12275 )
    9. JM109 E. coli competent cells (Promega, catalog number: L2001 )
    10. pSPL3 vector (Thermo Fisher Scientific, Invitrogen)
    11. Restriction endonuclease, XhoI (New England Biolabs, catalog number: R0146S )
    12. Restriction endonuclease, BamHI (New England Biolabs, catalog number: R0136S )
    13. QIAquick PCR Purification Kit (QIAGEN, catalog number: 28104 )
    14. T4 DNA ligase (New England Biolabs, catalog number: M0202S )
    15. SOC medium (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15544034 )
    16. Luria Bertani broth (Lennox) powder microbial growth medium (Sigma-Aldrich, catalog number: L3022 )
    17. Select Agar (Thermo Fisher Scientific, InvitrogenTM, catalog number: 30391023 )
    18. Carbenicillin, disodium salt (Dot Scientific, catalog number: DSC46000-5 )
    19. Glycerol (Sigma-Aldrich, catalog number: G5516 )
    20. QIAprep Spin Miniprep Kit (QIAGEN, catalog number: 27104 )
    21. Carbenicillin antibiotic, 1,000x (see Recipes)
    22. LB (Luria-Bertani) medium with carbenicillin antibiotic (see Recipes)
    23. LB agar with carbenicillin antibiotic plates (see Recipes)

  4. Mammalian cell culture
    1. Serological pipettes
      5 ml (Corning, Costar®, catalog number: 4487 )
      10 ml (Corning, Costar®, catalog number: 4488 )
      25 ml (Corning, Costar®, catalog number: 4489 )
    2. Transfer pipette, polyethylene, general purpose blood bank, bulb draw 1.9 ml, sterile (Sigma-Aldrich, catalog number: Z350699 )
    3. Pasteur glass pipettes, 5.75” (for aspiration) (VWR, catalog number: 14673-010 )
    4. 25 cm2 (T25) cell culture flasks with 0.2 μm vent caps (Corning, catalog number: 430639 )
    5. COS-7 mammalian cells (ATCC, catalog number: CRL-1651 )
    6. Dulbecco’s modified Eagle’s medium, DMEM, + GlutaMAX-1 cell culture medium (Thermo Fisher Scientific, GibcoTM, catalog number: 10569010 )
    7. Fetal bovine serum, FBS (Thermo Fisher Scientific, GibcoTM, catalog number: 10437028 )
    8. Phosphate-buffered saline, PBS, pH 7.4, 1x (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 )
    9. Penicillin-streptomycin, 10,000 U/ml (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
    10. Trypsin-EDTA (0.5%), no phenol red (Thermo Fisher Scientific, GibcoTM, catalog number: 15400054 )
    11. FuGENE 6 Transfection Reagent (Promega, catalog number: E2691 )
    12. Opti-MEM I reduced serum medium (Thermo Fisher Scientific, GibcoTM, catalog number: 31985062 )

  5. RNA extraction
    1. RNeasy Mini Kit (QIAGEN, catalog number: 74104 )
    2. QIAshredder (disposable cell-lysate homogenizers) (QIAGEN, catalog number: 79654 )

  6. cDNA generation
    1. High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4374966 )

  7. RT-PCR
    1. 0.2 ml PCR tubes with caps (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM12225 )
    2. HotStarTaq DNA polymerase (QIAGEN, catalog number: 203203 )
    3. TE buffer, 10 mM Tris 1 mM EDTA pH 8.0 (Sigma-Aldrich, catalog number: 93283 )
    4. Vector-specific oligonucleotide primers (Integrated DNA Technologies; https://www.idtdna.com/site/order/oligoentry):

      Note: Resuspend primers and make 10 μM stocks with TE buffer.

    5. 2 mM dNTPs (see Recipes)

  8. Sanger sequencing
    1. Sanger sequencing (GeneWiz commercial sequencing service; https://www.genewiz.com/en/Public/Services/Sanger-Sequencing)
    2. Sequencing primers (V1-F and V2-R oligonucleotide primers, same as for RT-PCR)


  1. Gel doc system (Labnet International, model: EnduroTM GDS )
  2. Water bath (37 °C) (Fisher Scientific, model: Fisher ScientificTM IsotempTM 215 )
  3. Bacterial shaking incubator (37 °C) (GeneMate, model: Incubated Shaker Mini )
  4. Bacterial incubator (37 °C) (Lab-Line Imperial III Incubator)
  5. Mammalian cell incubator (37 °C, 5% CO2) (Eppendorf, model: Galaxy® 170 S )
  6. Bunsen Burner, simple natural gas burner version (Fisher Scientific, catalog number: S95941 )
  7. NanoDrop Lite spectrophotometer (Thermo Fisher Scientific)
  8. Eppendorf centrifuge (benchtop) (Eppendorf, model: 5415 D )
  9. PCR thermocycler (Eppendorf, model: Mastercycler® Pro S )
  10. Pyrex bottle/flask, 500 ml
  11. HotPlate Stirrer (GeneMate, model: HotPlate Magnetic Stirrer )
  12. VWR Spinbar Magnetic Stir Bar, Polygon with Pivot Ring, 6 mm (¼") diameter, 35 mm (1⅜") length (VWR, catalog number: 74950-290 )
  13. Fisher Vortex Genie 2 (Thermo Fisher Scientific, catalog number: 12-812 )
  14. Household microwave oven, 1,000 W
  15. Gel electrophoresis tank (and tray) (Bio-Rad Laboratories, model: Wide Mini-Sub GT Cell )
  16. Electrophoresis power pack (Bio-Rad Laboratories, model: PowerPacTM Basic Power Supply )
  17. Autoclave (Medium Healthcare Sterilizer) (Tuttnauer, model: 6690 )
  18. Biological safety cabinet (The Baker Company, model: SterilGARD e3 High Efficiency SG403A-HE )
  19. Handheld aspirator and collector trap (Argos Technologies, model: EV514 )


  1. Sequencher software (version 5.2.4, Gene Codes Corporation; http://www.genecodes.com/sequencher)
  2. Primer3Plus (http://primer3plus.com/cgi-bin/dev/primer3plus.cgi)


  1. Amplification of experimental genomic fragment
    Custom oligonucleotide primers are designed to amplify the genomic regions of interest and incorporate restriction endonuclease sites at their 5’ ends, ready for subsequent cloning. An additional 8 bp is added to the 5’ end of each primer to increase the efficiency of post-PCR endonuclease digestion. Primer design is performed using Primer3Plus (http://primer3plus.com/cgi-bin/dev/primer3plus.cgi). For the first construct, modeling the variant within the 5’ splice donor site of exon 5 (c.760+2T>C), a 1,457 bp genomic fragment of the TEK gene encompassing all of intron 4, exon 5, intron 5, exon 6 and intron 6 was amplified. This gDNA sequence lacked recognition sites for both XhoI and BamHI endonucleases, so these two sites were incorporated into the primers for downstream cloning. For the second construct, modeling the variant within the 5’ splice donor site of exon 22 (c.3300+2delT), an 831 bp genomic fragment of the TEK gene encompassing all of intron 21, exon 22 and intron 22 was amplified, and XhoI and BamHI recognition sites were also incorporated for subsequent cloning. Phusion Hot Start II High-Fidelity DNA polymerase is used for amplification, due to its low error rates, fast extension speed and ability to handle larger amplicon sizes. Genomic DNA from both patient and control subjects should be used as template to amplify both mutant and wild-type genomic regions, respectively.
    Note: If the patient harbors a heterozygous mutation, both mutant and wild-type alleles can be sub-cloned from this single gDNA sample.
    1. In 0.2 ml PCR tubes, set up 20 μl PCR reactions as follows:

      Include a negative control sample (add 3 μl water instead of gDNA) to test for DNA contamination.
    2. Use the ‘Tm Calculator’ (www.thermofisher.com) to calculate the appropriate annealing temperature for PCR cycling. If the Tm values are higher than 69 °C, as was the case for the two TEK gene examples, a 2-step cycling protocol using a 72 °C combined annealing-extension step is recommended.
    3. PCR cycling should be performed as follows:

    4. Agarose gel electrophoresis should be performed as follows:
      1. Prepare a 1% agarose gel in TAE buffer containing SYBR safe DNA gel stain (see Recipes).
      2. Add 1 μl of 10x gel loading dye to 4 μl of water and 5 μl of the PCR reaction. Load all 10 μl into a well of the agarose gel. Add 5 μl of HyperLadder 1 kb marker to an adjacent lane of the gel for sizing.
      3. Perform agarose gel electrophoresis for approximately 30 min at 120 V.
      4. Inspect the gel using a UV lamp gel documentation system. A single distinct band should be present at the expected size (1,485 bp for the TEK exon 5 amplimer and 859 bp for the TEK exon 22 amplimer) before proceeding further (Figure 2).

        Figure 2. PCR amplification of genomic fragments. The TEK exon 5 (E5) and exon 22 (E22) amplimers are observed at 1,485 bp and 859 bp, respectively. –ve, PCR negative controls (water substituted for gDNA template) for each amplimer reaction; 1% agarose gel; molecular weight markers are 5 μl of ‘HyperLadder 1 kb’ per lane.

  2. Cloning of genomic fragments into pSPL3 exon trapping vector
    The PCR products are now purified and inserted into the multiple cloning site (MCS) of the exon trapping vector, pSPL3. The MCS is located within an intronic region between vector exons V1 and V2. First, the PCR products are purified to remove buffers, unincorporated dNTPs and primers. Then, the vector and PCR products are digested with the relevant restriction endonucleases, in these examples XhoI and BamHI, and again purified. The ‘sticky-ended’ vector and genomic ‘insert’ fragments are then ligated together. The ligation products are transformed into chemically competent JM109 E. coli cells and plated onto LB plates containing carbenicillin antibiotic for selection of plasmid-carrying bacteria. After incubating the plates overnight, several bacterial colonies are picked, grown up in liquid culture, and their plasmid content harvested. The purified plasmid DNA is then Sanger sequenced to verify which of the plasmid clones contains an insert with the correct content and orientation.
    1. Purify the PCR products using a QIAquick PCR Purification Kit column following QIAGEN’s instructions. This is the ‘insert’ DNA and should be at a concentration of 50-150 ng/μl. To achieve this concentration, it may be necessary to pool 3 x 20 μl PCR reactions into each purification column and to elute in a final volume of 30 μl elution buffer (EB).
    2. The pSPL3 plasmid DNA should also be pure in 1x TE or in EB from a QIAprep Spin Miniprep Kit purification. This is the ‘vector’ DNA and should be at a concentration of 200-400 ng/μl. If pSPL3 plasmid needs to be harvested from bacteria using a Miniprep Kit, please refer to step 7 of this section (‘Grow up and harvest plasmid from bacterial colonies’).
    3. Set up restriction double digestions in 0.5 ml PCR tubes as follows:

      Incubate reactions at 37 °C in a water bath for 2 h.
    4. Purify the digested DNA products using another QIAquick PCR Purification Kit column following QIAGEN’s instructions. This removes the buffer salts and unwanted small digestion fragments. Elute the insert sample in 30 μl EB and the vector sample in 50 μl EB. DNA concentrations will be approximately 50-70 ng/μl for both insert and vector. At this point, 5 μl of each sample can be run out on a 1% agarose gel to check fragments are of the expected sizes (1,463 bp for the TEK exon 5 insert fragment, 837 bp for TEK exon 22 insert fragment, and 6,011 bp for the digested pSPL3 vector fragment; Figure 3).

      Figure 3. Fragments of PCR products and pSPL3 vector following restriction digestion with BamHI and XhoI endonucleases. The TEK exon 5 (E5) and exon 22 (E22) fragments are observed at 1,463 bp and 837 bp, respectively. The pSPL3 vector (V) fragment is observed at 6,011 bp. A sample of undigested supercoiled pSPL3 vector (UV) is also included on the gel, migrating at ~4,000 bp. 1% agarose gel; molecular weight markers are 5 μl of ‘HyperLadder 1 kb’ per lane.

    5. Next, the insert and vector fragments will be ligated together in a 3:1 molar ratio. The NEBioCalculator tool (https://nebiocalculator.neb.com/) is helpful for calculating the amount of insert required to achieve these molar ratios. The linearized pSPL3 vector fragment is 6.011 kb, and the TEK exon 5 and exon 22 inserts are 1.463 kb and 0.837 kb, respectively. For 200 ng of vector DNA, 146 ng and 84 ng of the insert DNA will be required for each ligation, respectively. Set up ligation reactions in 0.2 ml PCR tubes as follows:

      1. Add the ligase enzyme last. ‘E5/E22’ refers to the TEK exon 5 and exon 22 inserts, respectively. The ‘vector only’ sample is a control to determine the degree to which vector DNA may be re-ligating to itself in the experimental ‘insert + vector’ sample.
      2. Set a PCR thermocycler to incubate the ligation reactions at 16 °C overnight (~16 h), followed by a heat-inactivation step of 65 °C for 10 min, then holding at 4 °C.
    6. Next, transform the ligation products into competent JM109 E. coli using a modified version of the Multiple-Use Protocol supplied by Promega (requires use of less cells):
      1. Heat a water bath to exactly 42 °C.
      2. For each sample, label and chill a 1.5 ml Eppendorf tube on ice.
      3. Thaw an aliquot of frozen competent cells on ice.
      4. When cells are just thawed (~10 min), gently flick the tube to mix the cells.
      5. Add 50 μl of cells to chilled Eppendorf.
      6. Add 25 ng (~5 μl) of DNA from ligation reaction to cells and gently flick to mix.
      7. Leave tube containing cell-DNA mix on ice for 10 min.
      8. Heat-shock cells for 50 sec in the 42 °C water bath and immediately return the tube to ice for 2 min.
      9. Add 450 μl of SOC medium (room temperature) and flick to mix.
      10. Secure tubes horizontally in an incubator for 1 h at 37 °C with shaking at 225 rpm.
      11. Make 1:10 and 1:100 dilutions of the respective transformed cells with SOC medium, as follows: To a clean 1.5 ml Eppendorf tube, add 450 μl of SOC medium and 50 μl of the transformed cell suspension and mix by gently flicking the tube–this is the 1:10 dilution. To another clean 1.5 ml Eppendorf tube, add 450 μl of SOC medium and 50 μl of the 1:10 cell dilution–this is the 1:100 dilution. Mix all three cell suspensions by gently flicking the tube immediately prior to plating the cells.
      12. On 3 LB plates containing 100 μg/ml carbenicillin, plate 100 μl of undiluted, 1:10 and 1:100 cell dilutions, respectively. Use a sterile cell spreader, or glass beads, to evenly distribute the cell suspension across each LB plate until all liquid is absorbed into the agar.
        Note: An effective cell spreader can be created by melting the end of a Pasteur glass pipette with a Bunsen burner flame so that it forms an ‘L’ shape. Sterilize the glass spreader between cell platings by immersing it into 100% ethanol and burning off the alcohol. Let the glass cool before spreading cells onto another plate.
      13. Incubate plates at 37 °C overnight.
      14. Examine plates for bacterial colonies. There should be more colonies on the ‘insert + vector’ plates than the ‘vector only’ plates (Figure 4).

        Figure 4. Bacterial colony growth on LB plates containing carbenicillin to select for circular pSPL3 plasmid-carrying bacteria. JM109 bacterial cells were transformed with either ‘insert + vector’ (panels A-C) or ‘vector only’ (panels D-F) ligation products and 100 μl was plated undiluted (panels A and D), at a 1:10 dilution (panels B and E), or at a 1:100 dilution (panels C and F).
        Note: No bacterial colonies were observed on the ‘vector only’ plates.

      15. The number of colonies on the ‘vector only’ plate will indicate the proportion of colonies on the ‘insert + vector’ plate that has resulted from vector re-ligation. This should be taken into consideration when deciding upon how many clones to pick in the following steps. If there are twice as many colonies on the ‘insert + vector’ plate compared to the ‘vector only’ plate, then half of the colonies on the ‘insert + vector’ plate are expected to have no insert present. Also, if the genomic DNA sample used to PCR amplify the insert sequence harbored a heterozygous mutation, half of the clones with an insert will contain wild-type sequence and half will contain the mutant sequence. If there are relatively few colonies on the ‘vector only’ plate, picking 5 colonies from the ‘insert + vector’ plate should suffice to obtain both wild-type and mutant insert-harboring plasmids.
    7. Grow up and harvest plasmid from several (5 to 10) bacterial colonies as follows:
      1. Label 50 ml Falcon tubes.
      2. To each tube add 10 ml of LB media containing carbenicillin (100 μg/ml final concentration).
      3. Using a sterile pipette tip, transfer a single bacterial colony from a plate to a liquid media tube.
        Note: Colonies will be spaced further apart on the serial dilution plates.
      4. Vortex the tubes briefly to transfer the bacteria from the submerged pipette tips into the liquid media.
      5. Place the tubes into a rack and incubate at 37 °C overnight with shaking at 225 rpm.
      6. Before harvesting the plasmid content, make glycerol stocks of the bacterial cultures for long-term storage:
        1. To a 2 ml cryovial, add:
          1) 500 μl 50% glycerol (diluted with dH2O).
          2) 500 μl overnight bacterial culture.
        2. Gently mix contents until homogeneous.
        3. Freeze at -80 °C (viable for years).
          Note: When recovering at a future date, scrape some of the frozen bacteria from the top of the stock and transfer to an LB plate containing antibiotic (thawing the stock is unnecessary and will reduce the lifetime of the sample).
      7. Harvest the plasmid content from the remainder of the overnight culture using a QIAprep Spin Miniprep Kit, following QIAGEN’s instructions.
      8. NanoDrop the plasmid DNA sample to quantify yield.
      9. Sanger sequence the purified plasmid DNA to verify which of the plasmid clones contains an insert with the correct content and orientation. Once validated, these are the final mini-gene constructs–a wild-type and mutant mini-gene for each splice variant being tested.

  3. Transfection of mini-gene constructs into COS-7 cells
    Next, COS-7 mammalian cells (adherent, fibroblast-like cells from the kidney of the African Green Monkey) are grown in culture and the wild-type and mutant mini-gene constructs are transfected into the cells using FuGene 6 transfection reagent following Promega’s instructions:
    1. COS-7 cells are seeded into 25 cm2 (T25) flasks at 25-40% confluence (6.25 x 105-1 x 106 cells) the day before transfection, so that they are 50-80% confluent the next day.
      Note: COS-7 cells have a doubling time of ~18 h.
    2. Prepare the transfection complex.
      Note: Include control transfections of only the pSPL3 vector with no gDNA insert, and another with no plasmid (QIAGEN buffer EB only).
      1. Allow FuGENE 6 and OptiMEM medium to equilibrate to room temperature.
      2. To a 1.5 ml Eppendorf tube, add:
        1. 475 μl OptiMEM medium
        2. 15 μl of FuGENE 6 (without touching the sides of tube with the tip, pipette into the center of the medium)
        3. Gently pipette up and down 10 times to mix
        4. Let stand for 5 min
        5. 5 μg (10 μl) of the respective wild-type or mutant pure plasmid DNA (at 500 ng/μl in QIAGEN buffer EB) or EB buffer alone in the control transfection (without touching the sides of tube with the tip, pipette into the center of the medium)
        6. Gently pipette up and down 10 times to mix
        7. Let stand for 15 min
    3. Dropwise, using a sterile plastic transfer pipette, add all 500 μl of the transfection mixture to the center of a T25 flask of cells, swirling gently with each drop added.
    4. Incubate the cells at 37 °C with 5% CO2 for 24 h.

  4. Total RNA isolation
    Next, total RNA is isolated from the COS-7 cells using an RNeasy Mini Kit following QIAGEN’s instructions:
    Note: A T25 cell culture flask containing COS-7 cells at 100% confluence (~2.5 x 106 cells) should yield ~80 μg RNA. Perform all steps including centrifugation at room temperature (21 °C). Add 10 μl 2-mercaptoethanol (2-ME) to each 1 ml of buffer RLT.
    1. Aspirate T25 flask culture medium and wash the cells in 5 ml PBS; aspirate all PBS.
    2. Add 3 ml trypsin-EDTA, swirl to coat the cells and quickly aspirate the solution.
    3. Incubate cells at 37 °C until they start to detach from the flask (~5 min).
    4. Add 5 ml medium containing 10% FBS to inactivate trypsin.
    5. Pipette the medium up and down within the flask to resuspend the cells and transfer the mixture to a 50 ml Falcon tube.
    6. Pellet the cells by centrifugation at 300 x g for 5 min; aspirate the supernatant.
    7. Add 350 μl buffer RLT (containing 2-ME), pipet to mix and add lysate directly into a QIAshredder spin column placed in a 2 ml collection tube.
    8. Centrifuge for 2 min at max speed in a benchtop centrifuge.
    9. Add 350 μl of 70% ethanol to the homogenized lysate; mix well by pipetting. Do not centrifuge at this point.
    10. Transfer up to 700 μl of sample (including any precipitate) to an RNeasy spin column placed in a 2 ml collection tube.
    11. Centrifuge for 15 sec at 9,000 x g; discard the flow-through.
    12. Add 700 μl buffer RW1 to the column.
    13. Centrifuge for 15 sec at 9,000 x g to wash the column membrane; discard the flow-through.
    14. Add 500 μl buffer RPE (containing ethanol) to the column.
    15. Centrifuge for 15 sec at 9,000 x g to wash the membrane; discard the flow-through.
    16. Add another 500 μl buffer RPE to the column.
    17. Centrifuge for 2 min at 9,000 x g to wash the membrane.
    18. Transfer the column to a new 2 ml collection tube and centrifuge at maximum speed for 1 min to remove any residual ethanol.
    19. Place the column in a new 1.5 ml collection tube.
    20. Add 50 μl RNase-free water directly to the membrane.
    21. Centrifuge for 1 min at 9,000 x g to elute the RNA sample.
    22. Add a further 30 μl RNase-free water directly to membrane.
    23. Centrifuge for 1 min at 9,000 x g to elute the remaining RNA.
    24. NanoDrop the RNA sample to quantify yield (expect ~70 μl total RNA, 400-700 ng/μl).
    25. Store the remaining RNA at -80 °C.

  5. cDNA generation
    Next, a High Capacity cDNA Reverse Transcription Kit is used with random primers to reverse-transcribe RNA into cDNA. To maximize the reaction, RNA samples should first be diluted to 200 ng/μl with RNase-free water. The reverse transcription reaction is then performed to convert 2 μg of total RNA to cDNA in a 20 μl volume, following Applied Biosystems’ instructions:
    1. On ice, for each sample, make 10 μl of a 2x RT Master mix:

    2. On ice, mix the components gently.
    3. Pipette 10 μl of the 2x RT Master mix into ice-cold 0.2 ml PCR tubes.
    4. Add 10 μl RNA (200 ng/μl) and gently flick to mix.
    5. Keep the reaction on ice until loaded into a PCR thermocycler programmed as follows:

    6. Store the resulting cDNA products at -20 °C.

  6. RT-PCR analysis of pSPL3-derived transcripts
    Next, the splicing vector-transcribed cDNA species are specifically amplified by PCR using primer pairs corresponding to sequences in the vector exons, V1 and V2 (Figures 1A and 1B). The resulting RT-PCR products from the wild-type and mutant mini-genes are visualized by gel electrophoresis and their sequence composition determined by Sanger sequencing (Figure 1C).
    Note: There is no need to remove the RNA component of the cDNA sample before PCR amplification. Use 4 μl of the cDNA sample as template in a 40 μl PCR reaction. More than 4 μl (10% of the PCR reaction volume) cannot be used, as carry-over of the buffer salts can inhibit the reaction.
    1. In 0.2 ml PCR tubes, set up 40 μl PCR reactions as follows:

      Note: Include a negative control sample (add 4 μl water instead of cDNA) to test for reaction contamination.
    2. Perform PCR amplification using the following cycling conditions:

    3. Analyze the PCR amplification products by electrophoresis on a 1% agarose gel (Figure 1C).
      Note: The pSPL3 vector with no gDNA insert will generate a single RT-PCR product of 257 bp from vector exons V1 and V2 (Figure 1C). Inclusion of TEK exons 5 and 6 will generate a single RT-PCR product of 530 bp (Figure 1C). Inclusion of TEK exon 22 will generate a single RT-PCR product of 357 bp (Figure 1C). From the mutant exon 5 and exon 22 splice site constructs, single bands of 551 bp and 357 bp are observed, respectively (Figure 1C).
    4. Where a single distinct band is present, the sample should be sent for direct Sanger sequencing using primers V1-F and V2-R (Figure 1C). If multiple bands are present, each band should be separately gel extracted (QIAquick Gel Extraction Kit) prior to Sanger sequencing.
      Note: From the mutant TEK exon 5 construct, Sanger sequencing determined the additional transcript content to result from inclusion of 21 bp of intron 5 (Figure 1C). From the mutant TEK exon 22 construct, Sanger sequencing showed a lack of any TEK content in the transcript, with content from vector exons V1 and V2 only, indicating exon 22 is skipped entirely during RNA splicing (Figure 1C).

Data analysis

Sequencher software (Gene Codes Corporation) can be used to analyze Sanger sequencing chromatogram files to determine the DNA sequence composition of the mini-gene transcripts.


  1. The Invitrogen website supplies an unconfirmed sequence of the 6,031 bp pSPL3 vector, assembled from the known sequence of fragments used to construct it: http://tools.thermofisher.com/content/sfs/vectors/pspl3_seq.htm.
  2. We re-sequenced our in-house pSPL3 vector (sequence given in Supplemental file 1) and found 3 bp lacking when compared to the sequence provided by Invitrogen. The 3 bases missing from our sequence were 2 bp in vector exon 1 and 1 bp in vector exon 2 (shown within square brackets). These sequence discrepancies are not proximal to exon-intron junctions and, as demonstrated by our experimental data, do not impact splicing mechanisms.
  3. In our 6,028 bp pSPL3 sequence (Supplemental file 1), the two vector exons are shown in bold and italics, with the V1-F and V2-R primers underlined. The multiple cloning site (MCS) recognition sequences for XhoI (CTCGAG) and BamHI (GGATCC) are also shown in bold and underlined.


  1. 2 mM dNTPs
    1. Thaw the 4 individual 100 mM dNTPs from the dNTP Set at room temperature
    2. Add 460 μl dH2O to a 1.5 ml screw-cap tube
    3. Add 10 μl of each dNTP
    4. Vortex to mix and store at -20 °C
  2. 1x TAE (Tris, acetate, EDTA) buffer
    40 mM Tris-HCl (pH 7.8)
    5 mM sodium acetate
    1 mM EDTA
  3. 1% or 1.5% agarose gel
    1. Add 1% or 1.5% (w/v) agarose to 1x TAE buffer in a 500 ml Pyrex bottle/flask
    2. Heat in a microwave oven, frequently swirling the bottle carefully until all agarose has been dissolved into solution
    3. Cool the agarose solution to ‘hand hot’ by flowing tap water over the side of the bottle whilst gently swirling
    4. Add 10 μl of SYBR safe DNA gel stain per 100 ml of gel solution and swirl to mix
    5. Immediately pour solution into an electrophoresis tray and allow to set at room temperature
  4. 10x gel loading dye
    1. To a 50 ml Pyrex bottle, add
      2.5 g Ficoll-400 (25% final conc.)
      1 ml of 1 M Tris-HCl (pH 7.8, 100 mM final conc.)
      2 ml 0.5 M EDTA, pH 8.0 (0.1 M final conc.)
      Make up to 10 ml with nuclease-free water
    2. Heat solution to 65 °C with a magnetic stir bar to dissolve Ficoll
    3. Add ~15 mg each of bromophenol blue and Orange G dyes (add up to 25 mg if a stronger dye intensity preferred)
    4. Mix well to dissolve dyes
    5. Store at room temperature
  5. Carbenicillin antibiotic, 1,000x
    1. Add 10 ml dH2O to a 50 ml Falcon tube
    2. Add 1 g carbenicillin disodium salt
    3. Vortex until salt has fully dissolved
    4. Sterilize the solution by passing it through a 0.2 μm-gage filter using a syringe
    5. Keep the 100 mg/ml stock solution frozen in 1 ml aliquots at -20 °C
  6. LB (Luria-Bertani) medium with carbenicillin antibiotic
    1. In a 1 L autoclavable bottle, add 20 g LB broth (Lennox) powder microbial growth medium to 1 L of dH2O
    2. Autoclave for 15 min at 121 °C to sterilize
    3. Allow to cool to room temperature before adding 1 μl per ml carbenicillin antibiotic
  7. LB agar with carbenicillin antibiotic plates
    1. Follow the recipe for LB medium, but add 15 g Select Agar before autoclaving
    2. Allow contents to cool to ‘hand-hot’ before adding 1 μl per ml carbenicillin antibiotic
    3. Pour ~25 ml into each plate (burst surface bubbles with a Bunsen burner flame)
    4. Allow to solidify at room temperature
    5. Bag plates upside-down in a stack and store at 4 °C until needed


This protocol was used for the work previously published in The Journal of Clinical Investigation (Souma et al., 2016). We thank Sean M. Martin for his careful reading of the manuscript and helpful comments. This study was funded by NIH R01 EY014685, the Research to Prevent Blindness Inc. Lew R. Wasserman Award, and the University of Wisconsin Centennial Scholars Award to Terri L. Young. The authors declare no conflict of interest.


  1. Church, D. M., Stotler, C. J., Rutter, J. L., Murrell, J. R., Trofatter, J. A. and Buckler, A. J. (1994). Isolation of genes from complex sources of mammalian genomic DNA using exon amplification. Nat Genet 6(1): 98-105.
  2. Duyk, G. M., Kim, S. W., Myers, R. M. and Cox, D. R. (1990). Exon trapping: a genetic screen to identify candidate transcribed sequences in cloned mammalian genomic DNA. Proc Natl Acad Sci U S A 87(22): 8995-8999.
  3. Nisson, P. E., Ally, A., Watkins, P. C. (1994). Protocols for trapping internal and 3’-terminal exons. PCR Methods Appl 4(1): S24-39.
  4. Souma, T., Tompson, S. W., Thomson, B. R., Siggs, O. M., Kizhatil, K., Yamaguchi, S., Feng, L., Limviphuvadh, V., Whisenhunt, K. N., Maurer-Stroh, S., Yanovitch, T. L., Kalaydjieva, L., Azmanov, D. N., Finzi, S., Mauri, L., Javadiyan, S., Souzeau, E., Zhou, T., Hewitt, A. W., Kloss, B., Burdon, K. P., Mackey, D. A., Allen, K. F., Ruddle, J. B., Lim, S. H., Rozen, S., Tran-Viet, K. N., Liu, X., John, S., Wiggs, J. L., Pasutto, F., Craig, J. E., Jin, J., Quaggin, S. E. and Young, T. L. (2016). Angiopoietin receptor TEK mutations underlie primary congenital glaucoma with variable expressivity. J Clin Invest 126(7): 2575-2587. 



背景 我们希望通过实验测试在TEK基因中鉴定的两个剪接供体位点变体c.760 + 2T> C和c.3300 + 2delT的功能影响(Souma等人,2016)。通常情况下,携带这些序列变体的个体不能获得细胞或mRNA的样品,因此我们利用外显子捕获方法作为功能测试。来自患者的DNA样品可用于感兴趣的基因组区域的PCR扩增。如果患者gDNA样品不可用,也可以通过诸如基于PCR的定点诱变等方法将序列变体并入野生型序列。
 外显子捕获方法最初是为了鉴定长期基因组DNA中的未知外显子而开发的(Duyk等人,1990)。创建了pSPL3外显子捕获载体以提高外显子鉴定的效率和可靠性,并且还允许筛选更大的基因组片段(Church et al。,1994; Nisson等,1994)。 pSPL3载体含有由SV40启动子组成的小型人造基因,具有功能性剪接供体和受体位点的外显子 - 内含子 - 外显子序列和晚期多聚腺苷酸化信号。在单个内含子内,定位多克隆位点,其中插入感兴趣的基因组片段以产生微型基因表达构建体。
 在我们的例子中,使用XhoI和BamHⅠ将来自TEK 基因的患者和对照基因组DNA片段进行PCR扩增并克隆在pSPL3载体外显子V1和V2之间 HI限制站点。然后用微型基因构建体转染COS-7细胞,并纯化得到的RNA含量。然后将mRNA转录物逆转录成cDNA。使用载体外显子特异性引物,从微型基因构建体产生的cDNA被特异性PCR扩增,并进行桑格测序。对于外显子5的5'剪接位点内的第一个剪接位点变体c.760 + 2T> C,包含所有内含子4,外显子5,内含子的TEK基因的1,457bp基因组片段5,外显子6和内含子6插入构建体(图1A)。微型基因表达转录物的RT-PCR和Sanger测序显示突变破坏了剪接供体位点,其在使用隐蔽剪接位点之前导致部分内含子5包含(图1C)。这种拼接错误被预测会导致翻译移码和提前终止信号,这可能导致通过废话介导的衰变途径的转录物消除。对于第二剪接位点变体,外显子22的5'剪接位点内的c.3300 + 2delT,包含所有内含子21,外显子22和内含子22的TEK基因的831bp基因组片段是插入结构(图1B)。微型基因表达转录物的RT-PCR和Sanger测序显示,剪接供体突变导致外显子22的跳跃,这也预示在患者的基因组背景中导致翻译移码和过早终止信号(图1C )。

图1.外显子捕获测定载体外显子V1和V2被描绘为黑盒子,而外显子5,6和22显示为灰色。载体外显子特异性引物由(A)和(B)中的半箭头表示。分别以构建体上方和下方的虚线表示野生型(WT)和突变体(M)剪接产物,其中包含碱基对的外显子大小。剪接位点突变的位置显示为星号(*)。使用含有TEK外显子5和6的野生型(WT-5)和突变型(M-5)基因组片段来建模c.760 + 2T> C突变。 B.使用含有外显子22的野生型(WT-22)和含有TEK外显子22的突变体(M-22)基因组片段来建模c.3300 + 2delT突变。 C.转染COS-7细胞的RT-PCR产物的凝胶电泳。 '空载体',用不含gDNA插入物的载体转染的细胞; 'TF -ve'(转染阴性),仅用QIAGEN缓冲液EB转染的细胞; “PCR -ve”(PCR阴性),PCR污染控制用水代替cDNA模板。通过Sanger测序确定的野生型和突变体转录物含量在凝胶图像的右侧描绘。显示在M5转录物内鉴定的额外的21bp内含子5序列,其在外显子5和6之间加入早熟终止密码子。

关键字:剪接, 突变, 外显子, 捕获, 表达, 转录, 变体, 剪接物


  1. PCR
    1. 0.2毫升具有盖帽的PCR管(Thermo Fisher Scientific,Invitrogen TM,目录号:AM12225)
    2. 1.5 ml锥形基座螺旋盖管(USA Scientific,目录号:1415-8399)
    3. 具有和不具有剪接位点变体(20ng /μl)的人类基因组DNA样品
    4. TE缓冲液,10mM Tris 1mM EDTA pH8.0(Sigma-Aldrich,目录号:93283)
    5. 定制合成的寡核苷酸引物将限制性内切酶位点引入5'末端(Integrated DNA Technologies; https://www.idtdna.com/site/order/oligoentry ):

        注意:基因组特异性序列加下划线,限制性位点以BOLD显示,并且向5'末端添加额外的8 bp显示为小写。 5'-CTCGAG-3'是XhoI的识别序列,5'-GGATCC-3'是BamHI的识别序列。 '-F'和'-R'分别指一对中的正向和反向引物。重悬引物,并用TE缓冲液制备10μM储备液

    6. Phusion热启动II高保真DNA聚合酶(Thermo Fisher Scientific,Thermo Scientific TM,目录号:F549S)
    7. 分子级水,无核酸酶(Dot Scientific,目录号:DS248700)
    8. dNTP集(QIAGEN,目录号:201913)
    9. 2 mM dNTPs(见配方)

  2. 凝胶电泳
    1. 1M Tris盐酸盐溶液,Tris-HCl,pH 7.8(Sigma-Aldrich,目录号:T2569)
    2. 3 M醋酸钠缓冲溶液,pH 5.2(Sigma-Aldrich,目录号:S7899)
    3. 0.5 M EDTA,pH 8.0(Santa Cruz Biotechnology,目录号:sc-203932)
    4. 琼脂糖(IBI Scientific,目录号:IB70042)
    5. SYBR安全DNA凝胶染色(Thermo Fisher Scientific,Invitrogen TM,目录号:S33102)
    6. Ficoll-400(Dot Scientific,目录号:DSF10400-25)
    7. 溴酚蓝,钠盐(MP Biomedicals,目录号:02152506)
    8. 橙G(Sigma-Aldrich,目录号:O3756)
    9. HyperLadder 1 kb(Bioline,目录号:BIO-33053)
    10. 1x TAE缓冲区(见配方)
    11. 1%或1.5%琼脂糖凝胶(参见食谱)
    12. 10x凝胶加载染料(参见食谱)

  3. 克隆
    1. 1.5ml Eppendorf管(VWR,目录号:20170-022)
    2. 培养皿,100 x 15毫米(VWR,目录号:25384-302)
    3. Whatman GD/X注射器过滤器,0.2μm孔径(Whatman,目录号:6901-2502)
    4. BD 60 ml注射器,Luer-Lok Tip(BD,目录号:309653)
    5. 巴斯德玻璃移液器,230毫米(用于在板上铺展细菌)(WHEATON,目录号:357335)
    6. 2 ml冷冻,自立(Simport,目录号:T310-2A)
    7. 50ml聚丙烯锥形管,Falcon(Corning,Falcon ®,目录号:352070)
    8. 0.5ml PCR管(Thermo Fisher Scientific,Invitrogen TM,目录号:AM12275)
    9. JM109大肠杆菌感受态细胞(Promega,目录号:L2001)
    10. pSPL3载体(Thermo Fisher Scientific,Invitrogen)
    11. 限制性内切核酸酶,Xho I(New England Biolabs,目录号:R0146S)
    12. 限制性内切核酸酶,Bam HI(New England Biolabs,目录号:R0136S)
    13. QIAquick PCR纯化试剂盒(QIAGEN,目录号:28104)
    14. T4 DNA连接酶(New England Biolabs,目录号:M0202S)
    15. SOC培养基(Thermo Fisher Scientific,Invitrogen TM,目录号:15544034)
    16. Luria Bertani肉汤(Lennox)粉末微生物生长培养基(Sigma-Aldrich,目录号:L3022)
    17. 选择Agar(Thermo Fisher Scientific,Invitrogen TM ,目录号:30391023)
    18. 碳青霉素,二钠盐(Dot Scientific,目录号:DSC46000-5)
    19. 甘油(Sigma-Aldrich,目录号:G5516)
    20. QIAprep Spin Miniprep Kit(QIAGEN,目录号:27104)
    21. 碳青霉素抗生素,1,000x(参见食谱)
    22. 含有羧苄西林抗生素的LB(Luria-Bertani)培养基(参见食谱)
    23. LB琼脂与羧苄青霉素抗生素板(见食谱)

  4. 哺乳动物细胞培养
    1. 血清移液管
      5 ml(Corning,Costar ®,目录号:4487)
      10 ml(Corning,Costar ®,目录号:4488)
      25 ml(Corning,Costar ®,目录号:4489)
    2. 转移移液管,聚乙烯,通用血库,灯泡抽取1.9 ml,无菌(Sigma-Aldrich,目录号:Z350699)
    3. 巴斯德玻璃移液器,5.75"(用于吸入)(VWR,目录号:14673-010)
    4. 具有0.2μm通气帽的25cm 2(T25)细胞培养瓶(Corning,目录号:430639)
    5. COS-7哺乳动物细胞(ATCC,目录号:CRL-1651)
    6. Dulbecco改良的Eagle's培养基,DMEM,+ GlutaMAX-1细胞培养基(Thermo Fisher Scientific,Gibco TM,目录号:10569010)
    7. 胎牛血清,FBS(Thermo Fisher Scientific,Gibco TM,目录号:10437028)
    8. 磷酸盐缓冲盐水,PBS,pH 7.4,1x(Thermo Fisher Scientific,Gibco TM,目录号:10010023)
    9. 青霉素 - 链霉素,10,000U/ml(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
    10. 胰蛋白酶-EDTA(0.5%),无酚红(Thermo Fisher Scientific,Gibco TM,目录号:15400054)
    11. FuGENE 6转染试剂(Promega,目录号:E2691)
    12. Opti-MEM I减少血清培养基(Thermo Fisher Scientific,Gibco TM,目录号:31985062)

  5. RNA提取
    1. RNeasy Mini Kit(QIAGEN,目录号:74104)
    2. QIAshredder(一次性细胞裂解物均质器)(QIAGEN,目录号:79654)

  6. cDNA生成
    1. 具有RNA酶抑制剂的高容量cDNA逆转录试剂盒(Thermo Fisher Scientific,Applied Biosystems TM,目录号:4374966)

  7. RT-PCR
    1. 0.2毫升具有盖帽的PCR管(Thermo Fisher Scientific,Invitrogen TM,目录号:AM12225)
    2. HotStarTaq DNA聚合酶(QIAGEN,目录号:203203)
    3. TE缓冲液,10mM Tris 1mM EDTA pH8.0(Sigma-Aldrich,目录号:93283)
    4. 载体特异性寡核苷酸引物(综合DNA技术; https://www。 idtdna.com/site/order/oligoentry ):


    5. 2 mM dNTPs(见配方)

  8. 桑格测序
    1. Sanger测序(GeneWiz商业测序服务; https://www.genewiz.com/en/Public/Services/Sanger-Sequencing
    2. 测序引物(V1-F和V2-R寡核苷酸引物,与RT-PCR相同)


  1. Gel doc系统(Labnet International,型号:Enduro TM GDS)
  2. 水浴(37℃)(Fisher Scientific,型号:Fisher Scientific TM Isotemp TM 215)
  3. 细菌摇床孵育器(37℃)(GeneMate,型号:Incubated Shaker Mini)
  4. 细菌培养箱(37℃)(Lab-Line Imperial III孵育器)
  5. 哺乳动物细胞培养箱(37℃,5%CO 2)(Eppendorf,型号:Galaxy 170S)
  6. 本森燃烧器,简单天然气燃烧器版本(Fisher Scientific,目录号:S95941)
  7. NanoDrop Lite分光光度计(Thermo Fisher Scientific)
  8. Eppendorf离心机(台式)(Eppendorf,型号:5415 D)
  9. PCR热循环仪(Eppendorf,型号:MastercyclerPro S)
  10. Pyrex瓶/瓶,500毫升
  11. HotPlate Stirrer(GeneMate,型号:HotPlate Magnetic Stirrer)
  12. VWR旋钮磁力搅拌棒,带有枢轴环的多边形,直径6 mm(¼"),长度35 mm(1"")(VWR,目录号:74950-290)
  13. Fisher Vortex Genie 2(Thermo Fisher Scientific,目录号:12-812)
  14. 家用微波炉,1000W
  15. 凝胶电泳槽(和托盘)(Bio-Rad Laboratories,型号:Wide Mini-Sub GT Cell)
  16. 电泳电源组(Bio-Rad Laboratories,型号:PowerPac TM 基本电源)
  17. 高压灭菌器(Medium Healthcare Sterilizer)(Tuttnauer,型号:6690)
  18. 生物安全柜(贝克公司,型号:SterilGARD e3高效SG403A-HE)
  19. 手持式吸气器和收集器阱(Argos Technologies,型号:EV514)


  1. Sequencher软件(版本5.2.4,Gene Codes Corporation; http://www.genecodes。 com/sequencher
  2. Primer3Plus( http://primer3plus.com/cgi-bin/dev/primer3plus.cgi


  1. 实验基因组片段的扩增
    设计定制的寡核苷酸引物以扩增感兴趣的基因组区域并在其5'末端掺入限制性内切核酸酶位点,随时进行克隆。每个引物的5'末端还加入8 bp,以提高PCR后内切酶消化的效率。引物设计使用Primer3Plus执行( http://primer3plus.com /cgi-bin/dev/primer3plus.cgi )。对于第一个构建体,对外显子5(c.760 + 2T> C)的5'剪接供体位点内的变体进行建模,包含所有内含子4的TEK基因的1,457bp基因组片段,外显子5,内含子5,外显子6和内含子6被扩增。该gDNA序列缺乏XhoI和Bam HI核酸内切酶的识别位点,因此将这两个位点并入引物用于下游克隆。对于第二个构建体,对外显子22(c.3300 + 2delT)的5'剪接供体位点内的变体进行建模,其中包含所有内含子21,外显子22的831bp的TEK基因组基因组片段并扩增内含子22,并且还引入XhoI和Bam HI识别位点用于随后的克隆。 Phusion热启动II高保真DNA聚合酶用于扩增,因为其错误率低,扩展速度快,处理较大扩增子大小的能力。应该使用来自患者和对照受试者的基因组DNA作为扩增突变体和野生型基因组区域的模板。
    1. 在0.2ml PCR管中,如下设置20μlPCR反应:

    2. 使用"T m Calculator"( www.thermofisher.com )计算适合的PCR循环退火温度。如果T m> 30%的值高于69℃,那么与两个TEK基因实例相同,使用72℃组合退火的2步循环方案推荐步骤。
    3. PCR循环应执行如下:

    4. 琼脂糖凝胶电泳应如下进行:
      1. 在含有SYBR安全DNA凝胶染色剂的TAE缓冲液中制备1%琼脂糖凝胶(参见食谱)
      2. 加入1μl10x凝胶加载染料至4μl水和5μlPCR反应液中。将所有10μl加入到琼脂糖凝胶的孔中。在凝胶的相邻泳道上加入5μl的HyperLadder 1kb标记。
      3. 在120V下进行约30分钟的琼脂糖凝胶电泳
      4. 使用UV灯凝胶记录系统检查凝胶。在继续进一步之前,应该以预期的大小(对于 TEK 外显子5扩增子为1,485bp)和 外显子22扩增子为859bp)存在单个不同的条带(图2 )。

        图2.基因组片段的PCR扩增分别在1,485bp和859bp分别观察到外显子5(E5)和外显子22(E22)扩增子。 -ve,每个扩增子反应的PCR阴性对照(水取代gDNA模板) 1%琼脂糖凝胶;分子量标记物是每泳道5μl'HyperLadder 1 kb'。

  2. 将基因组片段克隆到pSPL3外显子捕获载体中 PCR产物现在被纯化并插入到外显子捕获载体pSPL3的多克隆位点(MCS)中。 MCS位于载体外显子V1和V2之间的内含子区域内。首先,将PCR产物纯化以除去缓冲液,未掺入的dNTP和引物。然后,用相关的限制性内切酶消化载体和PCR产物,在这些实施例中,XhoI和BamHⅠ,再次纯化。然后将"粘性"载体和基因组"插入"片段连接在一起。连接产物转化成化学感受态的JM109E。大肠杆菌细胞并铺在含有羧苄青霉素抗生素的LB平板上,用于选择携带携带质粒的细菌。将板孵育过夜后,挑取几个细菌菌落,在液体培养物中长大,收获其质粒含量。然后将纯化的质粒DNA进行Sanger测序以验证哪个质粒克隆包含具有正确含量和取向的插入物。
    1. 按照QIAGEN的说明,使用QIAquick PCR Purification Kit纯化PCR产物。这是"插入"DNA,应该是浓度为50-150 ng /μl。为了达到这一浓度,可能需要将3×20μlPCR反应物合并到每个纯化柱中,并在终体积为30μl的洗脱缓冲液(EB)中洗脱。
    2. pSPL3质粒DNA也应在1x TE中纯化,或者来自QIAprep Spin Miniprep Kit纯化的EB。这是"载体"DNA,浓度应在200-400 ng /μl。如果需要使用Miniprep Kit从细菌中收获pSPL3质粒,请参阅本节的步骤7("从细菌菌落生长和收获质粒")。
    3. 在0.5ml PCR管中设置限制性双重消化,如下所示:

    4. 按照QIAGEN的说明,使用另一个QIAquick PCR Purification Kit纯化消化的DNA产物。这消除了缓冲盐和不需要的小消化片段。将插入样品在30μlEB中洗脱,并将载体样品洗脱至50μlEB。对于插入物和载体,DNA浓度将为约50-70ng /μl。在这一点上,每个样品可以在1%琼脂糖凝胶上流出5μl,以检查片段是否具有预期的大小(对于 TEK 外显子5插入片段为1,463bp,对于 > TEK 外显子22插入片段,对于消化的pSPL3载体片段为6,011bp;图3)。

      图3.使用Bam HI和XhoI核酸内切酶进行限制性消化后,PCR产物和pSPL3载体的片段。 分别在1,463bp和837bp分别观察到外显子5(E5)和外显子22(E22)片段。在6,011bp处观察到pSPL3载体(V)片段。未消化的超螺旋pSPL3载体(UV)样品也包含在凝胶上,迁移至约4,000bp。 1%琼脂糖凝胶;分子量标记物是每泳道5μl'HyperLadder 1 kb'。

    5. 接下来,插入片段和载体片段将以3:1的摩尔比连接在一起。 NEBioCalculator工具( https://nebiocalculator.neb.com /)对于计算实现这些摩尔比所需的插入量。线性化pSPL3载体片段为6.011kb,外显子5和外显子22插入片段分别为1.463kb和0.837kb。对于200ng载体DNA,分别需要146ng和84ng插入DNA。在0.2 ml PCR管中设置连接反应如下:

      1. 最后添加连接酶。 "E5/E22"分别是指TEK外显子5和外显子22插入片段。 "vector only"样本是一个对照,用于确定在实验"插入+载体"样本中载体DNA可能重新连接到其自身的程度。
      2. 设置PCR热循环仪,将连接反应在16℃温育过夜(〜16小时),然后在65℃下热灭活步骤10分钟,然后保持在4℃。
    6. 接下来,将结扎产物转化为有效的JM109E。大肠杆菌使用Promega提供的多用途协议的修改版本(需要使用较少的细胞):
      1. 将水浴加热至42°C。
      2. 对于每个样品,在冰上标记并冷却1.5 ml Eppendorf管。
      3. 在冰上解冻冷冻感受态细胞的等分试样。
      4. 当细胞刚解冻(〜10分钟)时,轻轻的轻轻摇动管子来混合细胞
      5. 将50μl细胞加入冷冻的Eppendorf。
      6. 从连接反应物中加入25ng(〜5μl)的DNA至细胞,轻轻摇动混合
      7. 将含有细胞DNA混合物的试管置于冰上10分钟
      8. 热休克细胞在42°C水浴中持续50秒,并立即将管返回冰中2分钟
      9. 加入450μlSOC培养基(室温),轻轻混匀
      10. 将培养箱在37℃水平放置1小时,并以225rpm摇动
      11. 用SOC培养基对各转化细胞进行1:10和1:100稀释,如下:向干净的1.5ml Eppendorf管中加入450μlSOC培养基和50μl转化细胞悬浮液,并轻轻摇动管这是1:10的稀释。向另一个干净的1.5ml Eppendorf管中加入450μlSOC培养基和50μl1:10细胞稀释液,这是1:100稀释。在电镀细胞之前,将所有三种细胞悬浮液轻轻轻轻地轻拍,直接将细管混匀
      12. 在含有100μg/ml羧苄青霉素的3 LB平板上,分别铺板100μl未稀释的,1:10和1:100的细胞稀释液。使用无菌细胞扩散器或玻璃珠,将细胞悬浮液均匀分布在每个LB平板上,直到所有液体被吸收到琼脂中。
      13. 孵育板37°C过夜。
      14. 检查细菌菌落的板块。在"插入+矢量"板上应该有比"仅矢量"板更多的殖民地(图4)

        图4.含有羧苄青霉素的LB平板上的细菌菌落生长用于选择携带环状pSPL3质粒的细菌 JM109细菌细胞用"插入+载体"(图片AC)或"仅载体"面板DF)连接产物和100μl未稀释(图A和D),1:10稀释(图B和E)或1:100稀释(图C和F)。

      15. "仅载体"板上的菌落数目将指示由载体重新连接导致的"插入+载体"平板上的菌落比例。在决定在以下步骤中挑选多少个克隆时,应该考虑这一点。如果与"仅载体"板相比,"插入+载体"板上的菌落数量是两倍,则"插入物+载体"板上的一半菌落预期不存在插入物。此外,如果用于PCR扩增插入序列的基因组DNA样品带有杂合突变,则具有插入片段的一半克隆将含有野生型序列,一半将含有突变序列。如果在"仅载体"板上存在相对较少的菌落,则从"插入+载体"平板中挑取5个菌落应足以获得野生型和突变型插入物质质粒。
    7. 从以下几个(5至10个)细菌菌落生长和收获质粒:
      1. 标记50毫升Falcon管。
      2. 向每个管中加入10ml含有羧苄青霉素(最终浓度为100μg/ml)的LB培养基
      3. 使用无菌移液器吸头,将单个细菌菌落从板转移到液体培养基管。
      4. 短暂旋转管道,将细菌从浸没式移液管尖端转移到液体介质中
      5. 将管放入架中,并在37℃下以225rpm摇动孵育过夜。
      6. 在收获质粒含量之前,将甘油储存的细菌培养物长期储存:
        1. 加入:
          1)500μl50%甘油(用dH 2 O稀释)。
        2. 轻轻混匀,直至均匀
        3. 在-80°C冷冻(多年可行)。
          注意:在将来的日期恢复时,从部分顶部刮掉一些冷冻的细菌,并转移到含有抗生素的LB板上(解冻不必要的库存,并减少样品的使用寿命)。 em>
      7. 按照QIAGEN的指示,使用QIAprep Spin Miniprep Kit从剩余的过夜培养物中收获质粒含量。
      8. NanoDrop将质粒DNA样品定量产量。
      9. Sanger对纯化的质粒DNA进行排序,以验证哪个质粒克隆含有正确含量和取向的插入片段。一旦验证,这些是最终的迷你基因构建体 - 每个待测试的剪接变体的野生型和突变型小基因。

  3. 将微型基因构建体转染到COS-7细胞中 接下来,COS-7哺乳动物细胞(来自非洲绿猴的肾脏的粘附的成纤维细胞样细胞)在培养物中生长,并且使用FuGene 6转染试剂将野生型和突变的小基因构建体转染到细胞中,Promega's说明:
    1. 将COS-7细胞以25-40%汇合(6.25×10 5/sup> -1×10 -6)接种到25cm 2(T25)烧瓶中>细胞),使其在第二天达到50-80%融合。
      注意:COS-7细胞的加倍时间为〜18 h。
    2. 准备转染复合物。
      1. 允许FuGENE 6和OptiMEM培养基平衡至室温
      2. 加入1.5ml Eppendorf管中
        1. 475μlOptiMEM培养基
        2. 15μl的FuGENE 6(不用接触管的两侧,将吸液管移入培养基的中心)
        3. 轻轻移液10次,以混合
        4. 放置5分钟
        5. 将5μg(10μl)各自的野生型或突变体纯质粒DNA(在QIAGEN缓冲液EB中为500ng /μl)或EB缓冲液中,单独在对照转染中(不接触管末端的吸管,移液至媒体中心)
        6. 轻轻移液10次,以混合
        7. 放下15分钟
    3. 使用无菌塑料转移移液管,将所有500μl转染混合物加入到T25瓶细胞的中心,每次滴加,轻轻旋转。
    4. 在37℃下用5%CO 2孵育细胞24小时。

  4. 总RNA分离
    接下来,使用RNeasy Mini Kit按照QIAGEN的说明书从COS-7细胞中分离总RNA:
    注意:含有100%汇合(〜2.5×10 6细胞)的COS-7细胞的T25细胞培养瓶应产生约80μgRNA。执行所有步骤,包括在室温(21°C)下离心。向每1ml缓冲液RLT中加入10μl2-巯基乙醇(2-ME)
    1. 吸出T25烧瓶培养基,并在5ml PBS中洗涤细胞;吸出所有PBS。
    2. 加入3ml胰蛋白酶-EDTA,旋转以涂覆细胞并快速吸出溶液
    3. 在37℃下孵育细胞,直到它们开始从烧瓶中分离(〜5分钟)
    4. 加入含有10%FBS的5ml培养基灭活胰蛋白酶
    5. 在培养瓶内上下移动培养基以重新悬浮细胞并将混合物转移到50ml Falcon管中。
    6. 通过在300×g离心5分钟来造粒细胞;吸出上清液。
    7. 加入350μl缓冲液RLT(含2-ME),移液管将混合溶液直接加入置于2 ml收集管中的QIAshredder旋转柱。
    8. 在台式离心机中以最大速度离心2分钟。
    9. 向匀浆的裂解液中加入350μl70%乙醇;通过移液混匀。此时不要离心。
    10. 将700μl样品(包括任何沉淀物)转移到置于2 ml收集管中的RNeasy旋转柱上。
    11. 以9,000 x g离心15秒;丢弃流通。
    12. 将700μl缓冲液RW1加入色谱柱。
    13. 以9,000×g离心15秒以洗涤柱膜;丢弃流通。
    14. 向柱中加入500μl缓冲液RPE(含乙醇)
    15. 以9,000 x g离心15秒以洗涤膜;丢弃流通。
    16. 添加另外500μl缓冲液RPE到列。
    17. 以9,000 x g离心2分钟以洗涤膜。
    18. 将柱转移到新的2ml收集管中,并以最大速度离心1分钟以除去任何残留的乙醇
    19. 将色谱柱放入新的1.5 ml收集管中。
    20. 将50μl无RNase的水直接加入膜中。
    21. 离心1分钟,以9,000×g/g的速度洗脱RNA样品。
    22. 再加入30μl无RNase的水直接加到膜上
    23. 以9,000 x g离心1分钟以洗脱剩余的RNA
    24. NanoDrop RNA样品以量化产量(预期〜70μl总RNA,400-700 ng /μl)。
    25. 将剩余的RNA储存在-80°C。

  5. cDNA生成
    接下来,使用高容量cDNA逆转录试剂盒与随机引物将RNA逆转录成cDNA。为了使反应最大化,RNA样品应首先用无RNase的水稀释至200 ng /μl。然后按照Applied Biosystems的说明书,进行逆转录反应以将2μg总RNA转化成20μl体积的cDNA。
    1. 在冰上,对于每个样品,制备10μl的2x RT Master混合物:

    2. 在冰上,轻轻混合组件。
    3. 将10μl的2×RT Master混合物吸入冰冷的0.2ml PCR管中
    4. 加入10μlRNA(200 ng /μl),轻轻摇动混匀
    5. 将反应保持在冰上,直到加载到PCR热循环仪中,程序如下:

    6. 将得到的cDNA产物储存在-20°C
  6. pSPL3衍生转录物的RT-PCR分析
    1. 在0.2ml PCR管中,如下设置40μlPCR反应:

    2. 使用以下循环条件进行PCR扩增:

    3. 通过在1%琼脂糖凝胶上电泳分析PCR扩增产物(图1C) 注意:没有gDNA插入的pSPL3载体将产生来自载体外显子V1和V2的257bp的单个RT-PCR产物(图1C)。包含TEK外显子5和6将产生530bp的单个RT-PCR产物(图1C)。包含TEK外显子22将产生357 bp的单一RT-PCR产物(图1C)。从突变外显子5和外显子22剪接位点构建体分别观察到551bp和357bp的单条带(图1C)。
    4. 如果存在单个不同的条带,则应使用引物V1-F和V2-R(图1C)将样品送至直接的Sanger测序。如果存在多个条带,则在Sanger测序之前,每个条带应单独凝胶提取(QIAquick Gel Extraction Kit)。
      注意:从突变体TEK外显子5构建体中,Sanger测序确定了由包含21bp内含子5而导致的额外的转录物含量(图1C)。从突变体TEK外显子22构建体中,Sanger测序显示转录本中缺少任何TEK含量,仅含有来自载体外显子V1和V2的内容,表明外显子22在RNA剪接期间完全跳过(图1C)。 br />


Sequencher软件(Gene Codes Corporation)可用于分析Sanger测序色谱图文件,以确定微型基因转录本的DNA序列组成。


  1. Invitrogen网站提供了一个未经确认的6,031 bp pSPL3载体序列,从已知的用于构建它的片段序列组装: http://tools.thermofisher.com/content/sfs/vectors/pspl3_seq.htm
  2. 我们重新排序了我们内部的pSPL3向量(在补充文件1 ),并且与Invitrogen提供的序列相比,发现3bp缺失。我们的序列缺失的3个碱基在载体外显子1中为2bp,载体外显子2中为1b(显示在方括号内)。这些序列差异不在外显子内含子结上,如我们的实验数据所示,不影响剪接机制。
  3. 在我们的6,028 bp pSPL3序列(补充文件1 ),两个载体外显子以粗体和斜体显示,V1-F和V2-R引物被下划线。 Xho I(CTCGAG)和Bam HI(GGATCC)的多重克隆位点(MCS)识别序列也以粗体显示,并加下划线。


  1. 2 mM dNTPs
    1. 在室温下从dNTP Set中解冻4个单独的100 mM dNTPs
    2. 向1.5 ml螺旋盖管中加入460μldH 2 O
    3. 加入10μl每个dNTP
    4. 涡旋混合储存于-20°C
  2. 1x TAE(Tris,乙酸盐,EDTA)缓冲液
    40mM Tris-HCl(pH7.8)
    1 mM EDTA
  3. 1%或1.5%琼脂糖凝胶
    1. 将1%或1.5%(w/v)琼脂糖加入到500ml Pyrex瓶/瓶中的1×TAE缓冲液中。
    2. 在微波炉中加热,经常旋转瓶子,直到所有琼脂糖溶解到溶液中
    3. 通过将自来水流过瓶子一侧,轻轻旋转,将琼脂糖溶液冷却"手热"
    4. 每100ml凝胶溶液加入10μlSYBR安全DNA凝胶染色液,旋转混匀
    5. 立即将溶液倒入电泳托盘,并允许在室温下放置
  4. 10倍凝胶加载染料
    1. 向50ml Pyrex瓶中加入
      1ml 1M Tris-HCl(pH 7.8,100mM最终浓度)
      2ml 0.5M EDTA,pH8.0(0.1M最终浓度)
    2. 用一个磁力搅拌棒将溶液加热至65°C,以溶解Ficoll
    3. 加入溴酚蓝和橙G染料约15mg(如果优选更强的染料强度,则加上25mg)
    4. 混合好以溶解染料
    5. 在室温下存放
  5. 羧苄青霉素抗生素,1,000倍
    1. 向10ml Falcon管中加入10ml dH 2 O
    2. 加入1μg羧苄青霉素二钠盐
    3. 漩涡直到盐完全溶解了
    4. 通过使用注射器
    5. 保持100毫克/毫升储备溶液冷冻1毫升等份在-20°C
  6. LB(Luria-Bertani)培养基与羧苄西林抗生素
    1. 在1L耐高压灭菌瓶中,加入20g LB肉汤(Lennox)粉末微生物生长培养基至1L dH 2 O O
    2. 在121℃高压灭菌15分钟以消毒
    3. 允许冷却至室温,然后加入1μl/ml羧苄青霉素抗生素
  7. LB琼脂与羧苄青霉素抗生素板
    1. 按照LB培养基的配方,但在高压灭菌前加入15 g选择Agar
    2. 在加入1μl/ml羧苄青霉素抗生素之前,让内容物冷却至"手热"
    3. 将每个板块倒入25毫升(本生灯火焰爆发的气泡)
    4. 允许在室温下固化
    5. 袋放置在堆叠中倒置并存放在4℃直到需要


该协议被用于以前在"临床调查杂志"(Souma等人,2016)发表的工作。我们感谢肖恩·马丁仔细阅读手稿和有用的意见。本研究由NIH R01 EY014685,"防止盲人研究"Lew R. Wasserman奖和威斯康星大学百周年纪念奖授予Terri L. Young的资助。作者宣称没有利益冲突。


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引用:Tompson, S. W. and Young, T. L. (2017). Assaying the Effects of Splice Site Variants by Exon Trapping in a Mammalian Cell Line. Bio-protocol 7(10): e2281. DOI: 10.21769/BioProtoc.2281.