Figure 1.
Assembly of RVDs into TALEN Entry plasmids and validation of TALEN function.
A. Repeat variable di-residues (RVDs, see Figure S1) are assembled by golden gate ligation into pTemp-S intermediate plasmids (pHex1-3), which are subsequently combined by golden gate ligation into the pENTR-TALEN entry vectors. No PCR amplification is required. TALEN entry vectors contain mCherry or EGFP reporter genes. B-D. Example of transfection of Ddx3x TALEN Entry plasmids into mouse Neuro-2a cells. B1-B2, Ddx3x left TALEN (with EGFP reporter); C1-C2; Ddx3x right TALEN (with mCherry reporter); D1-D2, Ddx3x TALEN pair. B1, C1, D1, Phase contrast; B2, C2, D2, epifluorescence. E. Surveyor mutation analysis of Ddx3x gene after transfection of Ddx3x TALENs vectors into Neuro-2a cells. F. Surveyor® mutation analysis of D1Pas1 gene after transfection of D1Pas1 TALENs vectors into Neuro-2a cells. RVD, repeat variable di-residues. EF, human EF1α promoter. CmR, Chloramphenicol resistance gene. ccdB, a lethal gene targeting DNA gyrase. FoKI, FoKI C-terminal domain. 2A, Thosea asigna virus 2A peptide. Spec+, Spectinomycin resistance gene.
Figure 2.
Transfer of TALEN sequences into adenoviral vectors and validation of TALEN function.
A. The left and right TALEN sequences described in Figure 1 were transferred into an adenoviral destination vector, pAd/PL-DEST, by Gateway® LR recombination. B-D, Example of transduction of Ddx3x TALEN adenoviruses into mouse Neuro-2A cells. B1-B2, left Ddx3x adenoviral-TALEN. C1-C2, right Ddx3x adenoviral-TALEN. D1-D2, Ddx3x adenoviral TALEN pair. E1-E2, Transduction of Ddx3y adenoviral-TALEN pair (both linked to mCherry) into male mouse fibroblasts. F1-F2, Transduction of Ddx3x adenoviral TALEN pair into mouse ES cells; left TALEN linked to EGFP and right TALEN linked to mCherry. G1-G2, Transduction of Ddx3y adenoviral-TALEN pair (both linked to mCherry) into rat C6 cells. H. Surveyor® mutation analysis of Ddx3x gene after transduction with Ddx3x adenoviral-TALENs into Neuro-2a cells. I. Surveyor® mutation analysis of Ddx3y gene after transduction of Ddx3y adenoviral-TALENs into male mouse fibroblasts.
Figure 3.
TALEN-induced mutations in mouse Ddx3 subfamily genes.
A. DNA sequence analysis of Ddx3x and D1Pas1 genes in Neuro 2A cells after transfection with plasmid TALENpairs. B. DNA sequence analysis of Ddx3y gene in male mouse fibroblasts after transduction with adenoviral-TALEN pairs. TALEN binding sequences are underlined. Stars indicate the identical sequences. Dashes indicate missing nucleotides compared to the wild type (WT) sequences. Exon 5 sequences of Ddx3y are in bold.
Figure 4.
Combination of a TALEN pair into one plasmid vector.
A. Left and right TALENs were ligated into different Entry plasmids to produce the “2 in 1” TALEN system. The left TALEN Entry vector contained attL1 and attR5 flanking sequences and the EGFP reporter. The right TALEN Entry vector contained attL5 and attL2 flanking sequences and the mCherry reporter. The left and right TALEN Entry vectors were then combined into the pPB-DEST destination vector by Gateway® LR recombination. In some cases Pgk-Puro was added to the destination vector to facilitate selection. B-D Example of transfection of Ddx3x TALEN plasmids into mouse Neuro-2a cells. B1 and B2: left Ddx3x TALEN Entry plasmid. C1 and C2, right Ddx3x TALEN Entry plasmid. D1 to D4, “2 in 1” Ddx3x TALEN plasmid. B1, C1, D1, phase contrast; B2, C2, D2, D3, D4, epifluorescence. E. Surveyor mutation analysis of the Ddx3x gene after transfection of the individual left (lane 1) or right (lane 2) Ddx3x TALEN Entry plasmids or the “2 in 1“ Ddx3x TALEN plasmid (lane 3) into Neuro-2a cells.