Daimon et al. have just published a paper in which they create and analyze a number of TALEN-knockout mutants that were JH-deficient or JH signaling-deficient.
While the biological findings reported in this paper are extremely interesting it is some of the technical aspects of the work that are worth noting here in Technology Topics.
Daimon et al. have used the analysis of mosaics to great advantage in this study and this could be useful for others working with insect systems other than Drosophila melanogaster were mosaic analysis is a well developed approach for conducting functional genomic studies.
Genes in which mutations result in lethality early in development pose problems for geneticists who would like to know if those genes play roles during other phases of development. This is a general problem in trying to fully understand the pleiotropy associated with genes. Mosaic analysis involving, say mitotic recombination, can enable cells with the appropriate null alleles to be created after early lethal critical periods, allowing phenotypes to be manifested later in development. In Drosophila somatic mosaics resulting from ‘natural’ mitotic recombination or now with more sophisticated methods resulting from FLP-mediated somatic recombination have been invaluable tools for fully probing gene function.
Daimon et al. created germ-line knockout mutations in JH acid methyltransferase (JHAMT) and the two Methoprene tolerant paralogs (Met1, Met2). Homozygotes resulted in early lethality.
Two types of mosaic analysis were performed to help them understand the roles of these genes in later stages of development.
For Met1 mosaic analysis they used the hereditary mosaic strain, mo. In the eggs of mo homozygotes the polar body nuclei do not degenerate in some eggs and the two nuclei in the egg are fertilized independently resulting in mosaic individuals. This is a mosaic analysis system that is unique to Bombyx.
The second mosaic analysis was used in their analysis of broad, a gene involved in specifying ‘pupa’. Their strategy in this case could be widely useful. Here they injected TALEN mRNAs into early embryos and then looked at the phenotypes of hatched larvae, pupae and adults. The authors found clones of cells with meaningful phenotypes and indicating that these cells had not become pupal or adult cells. 53 of the 87 pupae they examined had clones of cells that were presumably broad mutants.
The efficiency of TALEN mutagenesis in G0 embryos was high enough that the resulting larvae, pupa and adults could be used for phenotype analysis and no lines needed to be established. Highly active TALENs or Cas9 can result in high rates of conversion of both alleles of a gene resulting in clones of cells that are homozygous. This appears to be what Daimon et al. observed.
Others are also finding that TALEN and Cas9 mutagenesis is highly efficient. So much so that G0 individuals can be used to study phenotypes and make inferences about gene function
This approach could be generally useful for those working on ‘non-model’ insect systems and offers investigators with another approach besides gene silencing with dsRNA or undertaking the laborious and sometimes impossible task of creating germline mutations and establishing homozygous lines.
Daimon, T., Uchibori,M., Nakao, H., Sezutsu, H., and T. Shinoda (2015) Knockout silkworms reveal a dispensable role for juvenile hormone in holometabolous life cycle. PNAS published ahead of print July 20, 2015, DOI. 10.1073/pnas.1506645112