Rapid generation of splicing reporters with pSpliceExpress

Abstract

Almost all human protein-coding transcripts undergo pre-mRNA splicing and a majority of them is alternatively spliced. The most common technique used to analyze the regulation of an alternative exon is through reporter minigene constructs. However, their construction is time-consuming and is often complicated by the limited availability of appropriate restriction sites. Here, we report a fast and simple recombination-based method to generate splicing reporter genes, using a new vector, pSpliceExpress. The system allows generation of minigenes within one week. Minigenes generated with pSpliceExpress show the same regulation as displayed by conventionally cloned reporter constructs and provide an alternate avenue to study splice site selection in vivo.

Figure 1. Overview of the method

The minigene of interest can be cloned by two methods. Either a PCR product is directly cloned into pSpliceExpress (A, B, C) or it is first cloned into a gateway entry clone (D), which is then recombined with pDESTsplice (steps E–F) to generate the final construct (G). A. Amplification of the region of interest. Two primers F and R are used to amplify a part of the genomic DNA that harbors the alternative exon (black, splicing patterns are indicated). The primers have recombination sites that are indicated by circles. B. Construction of the splicing reporter using pSpliceExpress. The PCR fragment is recombined in vitro with pSpliceExpress vector. The vector contains Cm and ccdB selection markers that are used to isolate recombined clones. C. Structure of the final construct using pSpliceExpress. The inserted DNA is flanked by two constitutive rat insulin exons, indicated by doted pattern. The transcript is driven by a RSV LTR promoter (arrow) and the subcloned genomic fragment is flanked by attL sites, generated by the recombination of attB and attP sites. D. Subcloning of the genomic fragment for use with pDESTsplice. The genomic fragment of interest is generated with attB sites by PCR, which are recombined using any Gateway entry clone that has the ccdB-CmR selection cassette flanked by attL sites. E. Construction of the reporter gene using pDESTsplice. The attR1 sites of pDESTsplice are recombined in vitro with the attL1 sites of the entry clone. F. Structure of the final construct using pDESTsplice. The subcloned genomic fragment is flanked by attB1 sites. Except for the recombination sites, the structures of pDESTsplice minigenes are identical to those generated with pSpliceExpress (C). G. Analysis of the splicing reporter. The splicing reporter construct is transfected into a cell line of choice. The RNA generated is determined by RT-PCR, using the primers indicated (small arrows). The mRNA structures, indicated below the gene structure are expected to be generated by the construct.

Figure 2. Maps of the cloning vectors pDESTsplice and pSpliceExpress

A. pSpliceExpress. The vector contains the CmR, ccdB, colE1 ori, AmpR, SV40 ori and RS virus LTR that are indicated. Restriction sites of the multiple cloning sites are indicated. Two m13 sites can be used for sequencing. The selection cassette is flanked by attP sites. B. pDESTsplice. The vector has similar features as pSpliceExpress. The only difference is the attR attachment sites that are used for recombination.

Figure 3. Cloning efficiency of vectors used

The in vitro recombination typically generates more than 50 clones. The graphs show the percent of clones with an insert of the expected size, determined by PCR and restriction digest. Each point represents the percent of successful recombinations as a function of the insert length. A: Cloning efficiency of PCR products into pSpliceExpress B: Cloning efficiency between PCR fragments and pDONR221 C: Cloning efficiency between pDONR221 inserts and pDESTSplice.

Figure 4. Comparison between splicing reporters generated by pSpliceExpress and conventional cloning

A. Sequences for the primers used to generate the clk2 minigene. B. Structure of the pSpliceExpress generated clk2 reporter gene. The structure of the conventional cloned pclk2-Exontrap minigene is identical, except that BamHI and NotI restriction sites are present instead of the recombination sites. C. Cotransfection of tra2-beta1 with the conventionally cloned reporter gene. Numbers indicate µg transfected expression plasmid. A representative ethidium stained gels are shown. The structures of the RT-PCR products are indicated. A statistical evaluation of four independent experiments is shown below the gels. D. Cotransfection of tra2-beta1 with the clk2 reporter gene cloned by pSpliceExpress E. Cotransfection of CLK2 with the conventionally cloned reporter gene. F. Cotransfection of CLK2 with the clk2 reporter gene cloned by pSpliceExpress G. Structure of the pSpliceExpress generated 5-HTVaVbcons reporter gene. The structure of the conventional cloned 5HTVaVbcons-Exontrap minigene is identical, except that XhoI and BamHI restriction sites are present instead of the recombination sites. H. Cotransfection of ASF/SF2 with the conventionally cloned 5HTVaVbcons reporter gene. © denotes that the distal 5’ splice site of Exon V has been mutated to U1 binding consensus, which is described in (Kishore and Stamm, 2006). The star (*) indicates a heterodimer band. The statistical evaluation underneath the ethidium bromide stained gel represent the % exon Vb inclusion from three experiments. I. Cotransfection of ASF/SF2 with the 5HTVaVbcons reporter gene cloned by pSpliceExpress.

Figure 4. Comparison between splicing reporters generated by pSpliceExpress and conventional cloning

A. Sequences for the primers used to generate the clk2 minigene. B. Structure of the pSpliceExpress generated clk2 reporter gene. The structure of the conventional cloned pclk2-Exontrap minigene is identical, except that BamHI and NotI restriction sites are present instead of the recombination sites. C. Cotransfection of tra2-beta1 with the conventionally cloned reporter gene. Numbers indicate µg transfected expression plasmid. A representative ethidium stained gels are shown. The structures of the RT-PCR products are indicated. A statistical evaluation of four independent experiments is shown below the gels. D. Cotransfection of tra2-beta1 with the clk2 reporter gene cloned by pSpliceExpress E. Cotransfection of CLK2 with the conventionally cloned reporter gene. F. Cotransfection of CLK2 with the clk2 reporter gene cloned by pSpliceExpress G. Structure of the pSpliceExpress generated 5-HTVaVbcons reporter gene. The structure of the conventional cloned 5HTVaVbcons-Exontrap minigene is identical, except that XhoI and BamHI restriction sites are present instead of the recombination sites. H. Cotransfection of ASF/SF2 with the conventionally cloned 5HTVaVbcons reporter gene. © denotes that the distal 5’ splice site of Exon V has been mutated to U1 binding consensus, which is described in (Kishore and Stamm, 2006). The star (*) indicates a heterodimer band. The statistical evaluation underneath the ethidium bromide stained gel represent the % exon Vb inclusion from three experiments. I. Cotransfection of ASF/SF2 with the 5HTVaVbcons reporter gene cloned by pSpliceExpress.