Transgenic TALEN Expression to Control Sex in Bombyx

Emilie Pondeville, Ph.D., Institute of Infection, Immunity and Inflammation; University of Glasgow Centre for Virus Research

Emilie Pondeville, Ph.D., Institute of Infection, Immunity and Inflammation; University of Glasgow Centre for Virus Research

Analysis of gene function in insects by editing the genome, or reverse genetics, is now greatly facilitated thanks to powerful genetic technologies, such as TALENs, ZFNs, and more recently CRISPR/Cas9, becoming available in many organisms.

However, screening of transgenic mutated individuals is not easy mainly when the phenotype due to the mutation is not visible or only detectable in homozygotes. Moreover, if the mutated gene has toxic/lethal effects, maintaining transgenic lines can also be challenging for non-Drosophila insects as no balancer chromosome are available.

In this study, Xu et al (2014) decipher the function of the doublesex (dsx) gene in the Lepidoptera Bombyx mori. It is well known from Drosophila studies that dsx  is involved in sex determination and differentiation in insects. However, the hierarchy of sex determination gene function in Lepidoptera is poorly understood yet defining insect sex determination pathways is critical for developing genetic control technologies in Lepidoptera and other insects.  Indeed, male-only rearing has long been sought in sericulture in particular because males show superior economic characteristics, such as better fitness, lower food consumption, and higher silk yield (Nagaraju, 2002).

SILKWORM MOTHS MATING ON COCOONS. Image credit: Stephen Dalton—NHPA/EB Inc.

SILKWORM MOTHS MATING ON COCOONS.
Image credit: Stephen Dalton—NHPA/EB Inc.

 

To dissect dsx function during sex determination and differentiation throughout Bombyx development, Xu et al (2014) developed an elegant binary system using TALENs targeting Bmdsx female-specific exon 3.  In this sytem, left and right arms of the Bmdsx-specific binary TALEN are constitutively expressed in separate transgenic lines (respectively L and R lines) established by piggyBac-mediated transgenesis.  These lines were fully viable and fertile.  However,  crossing of the parental L and R lines results in widespread sequence-specific somatic mutagenesis  in the offspring resulting in 100% of the resulting females being sterile and having developmental defects.

SILKWORM MOTHS MATING ON COCOONS. Image Credit: Stephen Dalton—NHPA/EB Inc. The L line is marked with GFP and the R line is marked with dsRed2, both fluorochromes are directed by the eye-specific promoter 3XP3.

TRANSGENIC TALEN BINARY SYSTEM TO INDUCE SOMATIC EDITING AND STERILITY
The L line is marked with GFP and the R line is marked with dsRed2,
both fluorochromes are directed by the eye-specific promoter 3XP3.

Mutation of Bmdsx by somatic TALENs in females caused severely deleterious defects in ovary development, resulting in  females that are egg-free. Moreover, female Bmdsx mutation leads to abnormal external genitalia and copulation failure although all individuals still show normal sexual behavior. Overall, their data show that the products of Bmdsx play an important sex-specific role in sex determination and differentiation in Lepidoptera and demonstrate an interesting strategy for deploying TALENS.

Xu et al (2014) established a proof-of-concept of the usefulness of this new somatic TALEN binary system, which is highly efficient, particularly for analysis of genes without visible phenotypes or inducing toxic/lethal phenotypes. In addition, by using different promoters to regulate TALEN expression, this technique would allow one to dissect gene function according to sex, stage or tissue specificity.

Thus, this robust binary gene manipulation tool should be should be seriously considered for use in Lepidoptera as well as in other insect systems for functional genomic analysis and for the creation of genotypes useful for implementing insect genetic control strategies.
Xu J, Wang Y, Li Z, Ling L, Zeng B, James AA, Tan A, Huang Y (2014) Transcription activator-like effector nuclease (TALEN)-mediated female-specific sterility in the silkworm, Bombyx mori. Insect Mol Biol. doi: 10.1111/imb.12125.

 

Other Reference Cited:

Nagaraju J (2002) Application of genetic principles for improving silk production. Curr. Sci. 83 : pp. 409–414.

 

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