Efficient TALEN Construction for Bombyx mori Gene Targeting

Takasu, Yoko; Sajwan, Suresh; Daimon, Takaaki; Osanai-Futahashi, Mizuko; Uchino, Keiro; Sezutsu, Hideki; Tamura, Toshiki; Z̆urovec, Michal

doi:10.1371/journal.pone.0073458

Cited by 63 publications

(63 citation statements)

References 26 publications

(55 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In the mosaic analysis of broad, we injected TALEN mRNAs into embryos, and the hatched larvae were subjected to direct phenotypic analysis. Given that a number of genes have been targeted in Bombyx since the recent development of genome-editing tools (16,17), the former approach is very useful and could become a standard technique for functional studies of Bombyx genes. The latter approach facilitates the more rapid characterization of genes and in principle can be applied to a wide variety of animals.…”

Section: Discussionmentioning

confidence: 99%

“…To avoid these problems and to obtain definitive evidence to reveal the roles of JHs during the early larval instars, we performed knockout experiments targeted at genes involved in JH biosynthesis and JH reception in Bombyx, in which a highly efficient system for targeted mutagenesis mediated by transcription activator-like effector nucleases (TALENs) has been established (16,17). We generated a knockout mutant of JH acid methyltransferase (JHAMT) (18), which catalyzes the last step of JH biosynthesis in lepidopteran insects, as well as the two JH receptor genes in Bombyx, Met1, and Met2 (15).…”

Section: Significancementioning

confidence: 99%

See 1 more Smart Citation

Knockout silkworms reveal a dispensable role for juvenile hormones in holometabolous life cycle

Daimon

Uchibori

Nakao

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

121

132

View full text Add to dashboard Cite

Insect juvenile hormones (JHs) prevent precocious metamorphosis and allow larvae to undergo multiple rounds of status quo molts. However, the roles of JHs during the embryonic and very early larval stages have not been fully understood. We generated and characterized knockout silkworms (Bombyx mori) with null mutations in JH biosynthesis or JH receptor genes using genome-editing tools. We found that embryonic growth and morphogenesis are largely independent of JHs in Bombyx and that, even in the absence of JHs or JH signaling, pupal characters are not formed in first-or second-instar larvae, and precocious metamorphosis is induced after the second instar at the earliest. We also show by mosaic analysis that a pupal specifier gene broad, which is dramatically up-regulated in the late stage of the last larval instar, is essential for pupal commitment in the epidermis. Importantly, the mRNA expression level of broad, which is thought to be repressed by JHs, remained at very low basal levels during the early larval instars of JH-deficient or JH signaling-deficient knockouts. Therefore, our study suggests that the long-accepted paradigm that JHs maintain the juvenile status throughout larval life should be revised because the larval status can be maintained by a JHindependent mechanism in very early larval instars. We propose that the lack of competence for metamorphosis during the early larval stages may result from the absence of an unidentified broadinducing factor, i.e., a competence factor. juvenile hormone | metamorphosis | ecdysone | TALEN | Bombyx

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Significancementioning

confidence: 99%

Knockout silkworms reveal a dispensable role for juvenile hormones in holometabolous life cycle

Daimon

Uchibori

Nakao

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

121

132

View full text Add to dashboard Cite

show abstract

“…Both apt-like-TALEN left and apt-like-TALEN right did not have homologous sequences in the genome. TALEN messenger RNAs were prepared and microinjected into 64 p S embryos using standard protocols 57 . To mutagenise silkworms with this TALEN pair, TALEN left mRNA (200 ng ml À 1 ) and TALEN right mRNA (200 ng ml À 1 ) were co-injected as a mixture.…”

Section: Methodsmentioning

confidence: 99%

The transcription factor Apontic-like controls diverse colouration pattern in caterpillars

et al. 2014

Self Cite

View full text Add to dashboard Cite

Genetic polymorphisms underlie the convergent and divergent evolution of various phenotypes. Diverse colour patterns on caterpillars, which are ecologically important, are good models for understanding the molecular backgrounds of phenotypic diversity. Here we show that a single evolutionarily conserved gene apontic-like (apt-like) encoding for a putative transcription factor accounts for the silkworm p locus, which causes at least 15 different larval markings involved in branch-like markings and eye-spot formation. The expression of apt-like and melanin synthesis genes are upregulated in association with pigmented areas of marking mutants Striped (p S ) and normal ( þ p ) but not in the non-marking allele plain (p). Functional analyses, ectopic expression, RNAi and TALEN, demonstrate that apt-like causes melanin pigmentation in a cell-autonomous manner. These results suggest that variation in p alleles is caused by the differential expression of the gene apt-like which induces targeted elevation of gene expressions in the melanin synthesis pathway.

show abstract

“…TALEN knockout experiments were conducted as reported previously (Takasu et al, 2013). A manual search was performed for a TALEN target site for Bm-cardinal in a conserved region among p50T, C108, and pe e20 strains.…”

Section: Knockout Experiments Using Talenmentioning

confidence: 99%

Positional cloning of a Bombyx pink-eyed white egg locus reveals the major role of cardinal in ommochrome synthesis

Osanai-Futahashi

Futahashi

et al. 2015

Heredity

View full text Add to dashboard Cite

Ommochromes are major insect pigments involved in coloration of compound eyes, eggs, epidermis and wings. In the silkworm Bombyx mori, adult compound eyes and eggs contain a mixture of the ommochrome pigments such as ommin and xanthommatin. Here, we identified the gene involved in ommochrome biosynthesis by positional cloning of B. mori egg and eye color mutant pink-eyed white egg (pe). The recessive homozygote of pe has bright red eyes and white or pale pink eggs instead of a normal dark coloration due to the decrease of dark ommochrome pigments. By genetic linkage analysis, we narrowed down the pe-linked region to~258 kb, containing 17 predicted genes. RNA sequencing analyses showed that the expression of one candidate gene, the ortholog of Drosophila haem peroxidase cardinal, coincided with egg pigmentation timing, similar to other ommochrome-related genes such as Bm-scarlet and Bm-re. In two pe strains, a common missense mutation was found within a conserved motif of B. mori cardinal homolog (Bm-cardinal). RNA interference-mediated knockdown and transcription activatorlike effector nuclease (TALEN)-mediated knockout of the Bm-cardinal gene produced the same phenotype as pe in terms of egg, adult eye and larval epidermis coloration. A complementation test of the pe mutant with the TALEN-mediated Bm-cardinaldeficient strain showed that the mutant phenotype could not be rescued, indicating that Bm-cardinal is responsible for pe. Moreover, knockdown of the cardinal homolog in Tribolium castaneum also induced red compound eyes. Our results indicate that cardinal plays a major role in ommochrome synthesis of holometabolous insects.

show abstract

Efficient TALEN Construction for Bombyx mori Gene Targeting

Cited by 63 publications

References 26 publications

Knockout silkworms reveal a dispensable role for juvenile hormones in holometabolous life cycle

Knockout silkworms reveal a dispensable role for juvenile hormones in holometabolous life cycle

The transcription factor Apontic-like controls diverse colouration pattern in caterpillars

Positional cloning of a Bombyx pink-eyed white egg locus reveals the major role of cardinal in ommochrome synthesis

Contact Info

Product

Resources

About