Figure 1.
Schematic representation of plasmid constructs.
Plasmid pCS2-TAL, pBlue-TAL and pBLTC contain a central region of 34 a.a. repeats (vertical hatch) flanked by N- terminal and C-terminal TAL sequences of different lengths (solid rectangles). SP6 and T7 indicate the SP6 and T7 promoters (open arrowheads), respectively. Arrows represent the Fok I nuclease domain, gray rectangles the 3′ and 5′ UTRs and rectangles with angled hatch the sequences encoding nuclear localization signals.
Figure 2.
(A) Positions of TALEN targets in the BmBLOS2 gene. Open rectangles and numbers above represent exons and their length in bp. The filled box is the 5′- UTR. The sequence above the line indicates part of exon 2 with BLTC target sites and their names. The sequence below the line indicates the third exon with BLT-2 and BLTS targets. Bgl II restriction site is underlined. The c above the Bgl II site indicates polymorphism in w1-pnd strain. (B) Positions of TALEN targets in the Bm-re gene. Open rectangles and numbers above represent exons and their lengths in bp. Filled boxes are 5′-and 3′ UTRs. The sequence below the line indicates the ninth exon (capital letters). Bgl II restriction site is underlined.
Table 1.
Summary of TALENs used in this study.
Figure 3.
Comparison of somatic cell test and germline targeting experiments in silkworms.
(A) In the somatic cell test the mutagenized G0 embryos were used for determination of frequency of mutated alleles by DNA analysis. (B) In the BmBLOS2 experiment (using w1-pnd Bombyx strain) the G1 individuals were screened for an oily phenotype. In Bm-re (RE) mutagenesis (using pnd strain) the frequency of mutant alleles was tested in G1 individuals by DNA analysis.
Table 2.
Efficiency of TALENs in BmBLOS2 mutagenesis estimated by a B. mori somatic cell assay.
Figure 4.
Somatic and germline mutations of BmBLOS2 in silkworm larvae.
(A) Fifth instar G0 larvae containing mosaic areas of BmBLOS2 mutant tissue (B). Third instar G1 larvae with oily mutant phenotype (marked by arrowheads).
Table 3.
Efficiency of TALENs in BmBLOS2 mutagenesis evaluated by the oily phenotype.
Figure 5.
Correlation between the results from a somatic cell test and frequency of G1 mutants in B. mori based on data from mutagenesis with BLTS and BLTC and Bm-re TALENs.
The number of BLTS and BLTC mutants is underestimated due to a lower number of males detectable by phenotypic screening. Solid circles represent BLTS, BLT-2 and BLTC, whereas the open circle denotes the Bm-re TALEN.
Figure 6.
TALEN-induced insertions/deletions in BmBLOS2.
(A) Mutant alleles obtained from the somatic assay; (B) Mutant alleles obtained from G1 individuals. Sequences of mutant alleles are aligned with the respective wild-type sequences. TALEN target sites in the wild-type sequences are highlighted. The number of individuals carrying identical mutation is indicated to the right of each sequence.
Figure 7.
Mutagenesis of the Bm-re gene.
Embryos were photographed five days after Bm-re TALEN microinjection. (A) not injected control, (B) Bm-re mutant, (C, D) embryos injected with buffer, (E-H) G0 embryos injected with TALEN mRNA.
Figure 8.
Alignment of mutated Bm-re alleles obtained from sequence analysis of G0 and G1 individuals.
The number of individuals carrying identical mutations is indicated to the right of each sequence. TALEN target sites in the wild-type sequences are boxed. (A) Mutant alleles from the somatic assay; (B) G1 germline mutations.
Table 4.
Efficiency of TALENs in Bm-re mutagenesis.