Effective Gene Trapping Mediated by <em>Sleeping Beauty</em> Transposon

<div><p>Gene trapping is a high-throughput approach to elucidate gene functions by disrupting and recapitulating expression of genes in a target genome. A number of transposon-based gene-trapping systems are developed for mutagenesis in cells and model organisms, but there is still much room for the improvement of their efficiency in gene disruption and mutation. Herein, a gene-trapping system mediated by <em>Sleeping Beauty</em> (<em>SB</em>) transposon was developed by inclusion of three functional cassettes. The mutation cassette can abrogate the splice of trapped genes and terminate their translation. Once an endogenous gene is captured, the finding cassette independently drives the translation of reporter gene in HeLa cells and zebrafish embryos. The efficiency cassette controls the remobilization of integrated traps through inducible expression of <em>SB</em> gene. Analysis of transposon-genome junctions indicate that most of trap cassettes are integrated into an intron without an obvious 3′ bias. The transcription of trapped genes was abrogated by alternative splicing of the mutation cassette. In addition, integrated transposons can be induced to excise from their original insertion sites. Furthermore, the Cre/LoxP system was introduced to delete the efficiency cassette for stabilization of gene interruption and bio-safety. Thus, this gene-trap vector is an alternative and effective tool for the capture and disruption of endogenous genes <em>in vitro</em> and <em>in vivo</em>.</p> </div

Inducible activity of the tilapia <i>Hsp70</i> promoter (<i>TiHsp70</i>) at 37

<p>°C<b>.</b> (<b>A</b>) A novel gene-trap vector mediated by <i>Sleeping Beauty</i> transposon. IR/DR(L) and IR/DR(R), left and right inverted repeat/directed repeat of the SB transposon; SA, splice acceptor; IRES, internal ribosome entry site; Neo, kanamycin resistance gene; poly(A), poly(A) signal; TiHsp70, tilapia <i>Hsp70</i> promoter; SB11, SB11 transposase gene. (<b>B</b>) EGFP was used to monitor the inducible activity of <i>TiHsp70 in vitro</i> and <i>in vivo.</i> HeLa cells were transfected with pTiHsp70-EGFP at the density of about 80% confluence, treated in medium at 37°C for 1 h and recovered at 32°C for 2 h after transfection. Images were taken under a Nikon TE2000 fluorescent microscope and cell numbers in three fields of view were counted. Zebrafish embryos at one-cell stage were microinjected with pTiHsp70-EGFP. Injected embryos at 24 hpf were incubated in rearing water at 37°C for 1 h and then recovered at 28°C for 2 h. Low magnification fluorescent imaging of zebrafish embryos was performed on a SteReo Lumar V12 microscope form Zeiss and total embryos in three dishes were counted. (<b>C</b>) Statistical analysis of EGFP-expressing cells or embryos in (<b>B</b>). Data are given as means ± standard Deviation (n = 3). * indicate <i>P</i><0.05 versus the corresponding control. (<b>D</b>) Western blot analysis of EGFP in HeLa cells and zebrafish embryos. Heatshock-treated and -untreated cells (transfected with pTiHsp70-EGFP) and embryos (injected with pTiHsp70-EGFP) samples were undergone western blot and the pEGFP-F plasmid was used as a positive control. The expression of reference gene <i>β-actin</i> was also analyzed in these samples.</p

Molecular analysis of the trapped gene <i>mamdc2a</i> in transgenic zebrafish.

<p>(<b>A</b>) Schematic representation of the insertion of gene trap cassette in the intron7 of gene. The locations of primers used for molecular analysis are indicated. (<b>B</b>) Integrity analysis of gene trap cassette in transgenic fish. Genomic DNA was extracted from the trail of transgenic fish and subjected to PCR analysis with primes indicated in (<b>A</b>). (<b>C</b>) RT-PCR analysis of <i>mamdc2a</i> transcript in wild-type (+/+), heterozygous (+/−) and homozygous (−/−). Primers E7F/E8R were used to amplify the endogenous transcript and primers E7F/R3 were used to amplify the exon7-EGFP fusion transcript. The <i>β-actin</i> transcript was amplified as an internal control. (<b>D</b>) The fusion transcript generated from the proper splicing of EGFP gene and its upstream exon in <i>mamdc2a</i> gene. Sequences in red represent exon7 of <i>mamdc2a</i> gene. Sequences in yellow represent the SA signal. Sequences in gray indicate the IRES followed by the partial EGFP sequence in green. (<b>E</b>) qRT-PCR was performed with primers E7F/E8R to determine the expression of endogenous <i>mamdc2a</i> gene in wild-type, heterozygous and homozygous embryos at 72 hpf. The <i>mamdc2a</i> levels were normalized to the <i>β-actin</i> mRNA levels. (<b>F</b>) qRT-PCR analysis of SB11 expression in 24 hpf embryos from <i>mamdc2a</i>-transgenic line after heat shock at 37°C for 3 h and recovery at 28°C for 2 h. (<b>G</b>) Detection of Cre-mediated TiHsp-SB11 excision by southern hybridization. Cre reombinase mRNA was microinjected into wild-type, heterozygous and homozygous embryos. “+” represents the presence of Cre recombinase and “−” represents the absence of Cre recombinase. Line1–2: DNA from wild-type. Line 3–4: DNA from heterozygous. Line 5–6: DNA from homozygous.</p

Activity of the trapping cassette in an exon of pSPL3-E3 vector.

<p>pSPL3-E3 was generated by insertion of an exon from carp beta-actin gene (Exon3). The trapping cassette was then sucloned into the <i>Bam</i>HI/<i>Eco</i>RI site of pSPL3-E3 vector to generate the pSPL3-E3/Trap(exon), which was used for transient transfection of HeLa cells at 80% confluence. Images were taken under a Nikon TE2000 fluorescent microscope at 48 h after transfection and cell numbers in three independent transfections were counted. Zebrafish embryos at one-cell stage were microinjected with the pSPL3-E3/Trap(exon). Injected embryos at 24 hpf were imaged under a SteReo Lumar V12 microscope form Zeiss and total embryos in three dishes were counted. The ectopic expression of EGFP in one embryo was enlarged and shown in a merged image. SD1, splice donor for exon1; SA3, splice acceptor for exon3; IR/DR(L) and IR/DR(R), left and right inverted repeat/directed repeat of the SB transposon; SA, splice acceptor; IRES, internal ribosome entry site; EGFP, enhanced green fluorescence protein gene; poly(A), poly(A) signal; SD3, splice donor for exon3; SA2, splice acceptor for exon2.</p

Integration of the SB-based gene trap efficiently disrupts the expression endogenous genes in HeLa cells.

<p>(<b>A</b>) Schematic representation of a gene trapping insertion in an endogenous gene and potential transcripts in Hela cells. Endogenous exons are boxed and arrows indicate the positions of primers used for transcript analysis. IR/DR(L) and IR/DR(R), left and right inverted repeat/directed repeat of the SB transposon; SA, splice acceptor; IRES, internal ribosome entry site; Neo, kanamycin resistance gene; poly(A), poly(A) signal; TiHSP70, tilapia <i>Hsp70</i> promotor; SB11, SB11 transposase gene. (<b>B</b>) RT-PCR analysis of transcripts from a trapped endogenous gene (<i>INPP5B</i>, <i>HERC2</i>, <i>TRIO</i> or <i>CPZ</i>) in four cell colonies. N: Normal HeLa cells; C: cell colonies; ET: Endogenous transcript; FT: Fusion transcript. The <i>GAPDH</i> is used as the control for equal amount of cDNA template in PCR reactions. (<b>C</b>) Assessing the mutagenicity of gene-trap insertions by qRT-PCR. Total RNA was isolated from each cell colony and subjected to qRT-PCR analysis. The mRNA expression levels of an endogenous gene in the normal Hela cells and two other colonies without insertion at the gene loci (the controls) were compared to that in a cell colony containing an insertion at the corresponding gene loci. N represent normal HeLa cells; C1 represents <i>INPP5B</i> gene; C2 represents <i>HERC2</i> gene; C3 represents <i>TRIO</i> gene; C4 represents <i>CPZ</i> gene. Data are given as Means ± Standard Deviation (n ≥3). ** and * indicate <i>P</i><0.01 and <i>P</i><0.05 versus the controls, respectively.</p

An IRES sequence is required for independent expression of the reporter gene.

<p>(<b>A</b>) The ECMV/IRES element in the pSPL3-Trap(intron) vector was deleted to generate the pSPL3-Trap(intron)ΔIRES. SD1, splice donor for exon1; IR/DR(L) and IR/DR(R), left and right inverted repeat/directed repeat of the SB transposon; SA, splice acceptor; IRES, internal ribosome entry site; EGFP, enhanced green fluorescence protein gene; poly(A), poly(A) signal; SA2, splice acceptor for exon2. (<b>B</b>) pSPL3-Trap(intron) and pSPL3-Trap(intron)ΔIRES constructs were transfected into HeLa cells at 80% confluence, respectively. Images were taken under a Nikon TE2000 fluorescent microscope at 48 h after transfection and cell numbers in three independent transfections were counted. (<b>C</b>) Zebrafish embryos at one-cell stage were microinjected with the pSPL3-E3/Trap(exon). Injected embryos at 24 hpf were imaged under a SteReo Lumar V12 microscope form Zeiss and total embryos in three dishes were counted. (<b>D</b>) Statistical analysis of EGFP-expressing cells in (<b>B</b>) or embryos in (<b>C</b>). Each construct was tested three times and each experiment was done in triplicate. Data are given as means ± standard Deviation. ** indicate <i>P</i><0.01 versus the corresponding control.</p

Activity of the trapping cassette in an intron of pSPL3 vector.

<p>The trapping cassette was subcloned into the <i>Bam</i>HI/<i>Eco</i>RI site of pSPL3 vector to generate the pSPL3-Trap(intron), which was used for transient transfection of HeLa cells at 80% confluence. Images were taken under a Nikon TE2000 fluorescent microscope at 48 h after transfection and cell numbers from three independent transfections were counted. Zebrafish embryos at one-cell stage were microinjected with the pSPL3-Trap(intron). Injected embryos at 24 hpf were imaged under a SteReo Lumar V12 microscope form Zeiss and total embryos in three dishes were counted. The ectopic expression of EGFP in one embryo was enlarged and shown in a merged image. SD1, splice donor for exon1; IR/DR(L) and IR/DR(R), left and right inverted repeat/directed repeat of the SB transposon; SA, splice acceptor; IRES, internal ribosome entry site; EGFP, enhanced green fluorescence protein gene; poly(A), poly(A) signal; SA2, splice acceptor for exon2.</p

Remobilization of trap inserts in the HeLa cell genome.

<p>(<b>A</b>) A schematic diagram of trap cassette remobilization and a canonical footprint left in the original insertion site. Arrows indicate the primer containing the footprint (F) and a gene-specific primer (R). IR/DR(L) and IR/DR(R), left and right inverted repeat/directed repeat of the SB transposon; SA, splice acceptor; IRES, internal ribosome entry site; Neo, kanamycin resistance gene; poly(A), poly(A) signal; TiHSP70, tilapia <i>Hsp70</i> promoter; SB11, SB11 transposase gene. (<b>B</b>) Individual cell colony containing a trap insertion at shown gene loci (<i>INPP5B</i>, <i>HERC2</i>, <i>GRXCR1</i>, <i>CACNA1E</i> or <i>RP11</i>) was cultured at 32°C in two 35 mm dishes wells. Cells on one dish (T) was subjected to heat induction at 37°C and cells on another dish (N) was kept at 32°C. Total genome DNA was isolated and subjected to PCR analysis using gene-specific primers (F+R) in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044123#pone.0044123.s003" target="_blank">Table S1</a>. (<b>C</b>) Sequencing trace files of independent remobilization events in cell colonies. These sequencing trace files representing independent remobilization event in genes named <i>INPP5B</i>, <i>GRXCR1</i> and <i>CACNA1E.</i> After the excision and remobilization of the gene-trap vector from the original insertion genome locus, footprint was generated as shown in bold box. (<b>D</b>) Southern hybridizations. Neo probes were used to detect the copy number of transposons in the genome of HeLa cells incubated at 32°C (-) and the gene-trap cassettes excised from their insertion sites after heat shock (+) at 37°C for 48 h and recovered at 32°C for 5 days. Arrows point to newly-generated inserts after heat shock. (<b>E</b>) Southern blotting analysis with Neo probes indicated that the size of genomic DNA fragments with the trap cassette (dashed arrows) reduced by the transient expression of the Cre recombinase (+).</p

Transcriptional analysis of the trapping cassette in an intron of pSPL3 vector.

<p>(<b>A</b>) Potential splice variants I and II from the pSPL3-Trap(intron). Sequencing trace files representing the splicing of SD1 with SA and SA1 were shown, respectively. SD1 aSD1, splice donor for exon1; IR/DR(L) and IR/DR(R), left and right inverted repeat/directed repeat of the SB transposon; SA, splice acceptor; IRES, internal ribosome entry site; EGFP, enhanced green fluorescence protein gene; poly(A), poly(A) signal; SA2, splice acceptor for exon2. (<b>B</b>) RT-PCR analysis of transcripts from pSPL3-Trap(intron)-transfected HeLa cells. Sequencing results indicate the 1.2-kb band is derived from the splice variant I, which contains exon1, IRES and EGFP, and the 270 bp band results from splice variant II, which represents the proper spicing of exon1 with exon2 in pSPL3. (<b>C</b>) The absolute quantification of cDNA using real-time PCR was employed to determine the copy numbers of transcripts including EGFP (F+R, E = 96.1%, R² = 0.9981) and exon2 (F+R1, E = 97.2%, R² = 0.9989). Data are given as means ±standard deviation (n = 3). ** indicate <i>P</i><0.01 versus the exon2 expression level in pSPL3-Trap(intron)-transfected cells.</p

Transcriptional analysis of the trapping cassette in an exon of pSPL3-E3 vector.

<p>(<b>A</b>) Potential splice variants I+III, II+III and IV from the pSPL3-E3/Trap(exon). SD1, splice donor for exon1; SA3, splice acceptor for exon3; IR/DR(L) and IR/DR(R), left and right inverted repeat/directed repeat of the SB transposon; SA, splice acceptor; IRES, internal ribosome entry site; EGFP, enhanced green fluorescence protein gene; poly(A), poly(A) signal; SD3, splice donor for exon3; SA2, splice acceptor for exon2. (<b>B</b>) RT-PCR analysis of transcripts from pSPL3-E3/Trap(exon)-transfected HeLa cells. Sequencing results indicate the 2421 bp and 1950 bp bands are derived from the splice variant I+III, the 1971 bp and 1500 bp bands from the splice variant II+III, and the 270 bp band from splice variant IV. The proper splicing of transcripts from pSPL3-E3 gives rise to a 411 bp band containing exon1, exon3 and exon2, and the 270 bp band containing exon1 and exon2. (<b>C</b>) The absolute quantification of cDNA using real-time PCR was employed to determine the copy numbers of transcripts including EGFP (F+R, E = 98.1%, R² = 0.9994) and exon2 (F+R1, E = 97.6%, R² = 0.9976). Data are given as means ±standard deviation (n = 3). ** indicate <i>P</i><0.01 versus the exon2 expression level in pSPL3-E3/Trap(exon)-transfected cells.</p