Crickets, and Gryllus bimaculatus in particular, are the subjects of research of neurobiologists, developmental biologist and others. Genetic technologies to foster the functional genomic analysis of these insects are in high demand even though these insects have a relatively long generation time (~ 2 months).
In 2012 Watanabe et al. describe their successful use of ZFNs and TALENs to create genetically modified G. bimaculatus. Here Watanabe et. al. (2014) report the detailed method they used for microinjecting TALENs into the embryos of G. bimaculatus, and a protocol for screening for mutants homozygous for a specific gene-knockout.
Watanabe et al (2012) were the first to report using TALENs (and ZFNs) to generate mutants in a hemimetabolous insect. The strategy used by Watanabe et al (2012) is described in detail in their current manuscript (Watanabe, et al. 2014) and could be widely useful to those working with other insects.
Eggs were collected for approximately 1 hr and allowed to age for an additional hour. The eggs were soaked briefly in 70% EtOH, rinsed in fresh water and aligned for injections. Chorions were not removed.
The washed embryos were collected and held on a wet a paper towel from which individual embryos were lifted and placed on double stick tape in a plastic well created by a plastic frame on top of the injection slide. Eggs were aligned such that their long axis was perpendicular to the needle. Once aligned the embryos were covered with mineral oil and held in a moist chamber until injected.
The TALEN-cocktail was injected into the dorsal side of embryos at a position approximately 20-25% of the embryos length from the posterior end. Injections were not made precisely at the posterior end of the egg as is often done with eggs from Diptera, for example.
Injection of the TALEN cocktail was accomplished by applying weak constant pressure, which aided in the prevention of back-filling of the needle during penetration of the egg. The precise delivery 2-4 nL of TALEN mRNA during injection was observed as a small bubble of solution within the embryo.
After all embryos were injected they were held for 2 days at 28ºC. On the second day the embryos were removed from the tape while at the same time removing as much mineral oil as possible with lab wipes. Embryos were transferred to humid Petri dishes and held under normal rearing conditions until hatch.
Seven days post injection 10 eggs were sacrificed to test for TALEN activity in the embryonic soma. Genomic DNA was extracted from these embryos and used as a PCR template for SURVEYOR nuclease treatment-based analysis for TALEN activity. From these results the activity of the injected TALEN could be assessed.
G0 nymphs were separated by sex and out-crossed to avoid mating between mutants. A sample of the offspring from this cross was again screened using the SURVEYOR PCR assay to identify which G0 crosses were producing mutants. Nymphs from crosses producing mutants were genotyped by isolating DNA from the tip of their hind legs. Heterozygous mutants identified in this way would then be crossed to produce progeny, some of which are expected to be homozygous if the mutant was not homozygous-lethal.
In Watanabe et al (2012) the authors report that 17% of the injected embryos developed into adults that produced mutagenized progeny. They were unable to create homozygotes in that experiment because homozygous mutants at their target locus (laccase) were lethal.
The detailed method reported by Watanabe et al. (2014) provides a good framework from which others can plan TALEN mutagenesis of “their” species.
Watanabe T, Noji S, Mito T (2014) Gene knockout by targeted mutagenesis in a hemimetabolous insect, the two-spotted cricket Gryllus bimaculatus, using TALENs. Methods 69: 17-21 DOI: 10.1016/j.ymeth.2014.05.006
Other Reference Cited:
Watanabe T, Ochiai H, Sakuma T, Horch HW, Hamaguchi N, Nakamura T, Bando T, Ohuchi H, Yamamoto T, Noji S, Mito T (2012) Non-transgenic genome modifications in a hemimetabolous insect using zinc-finger and TAL effector nucleases. Nat Commun 3 DOI; 10.1038/ncomms2020
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