Megaselia and Chironomus Germline Transformation

hassan

Hassan M. Ahmed, Graduate student in Developmental Biology, Georg-August-Universität Göttingen
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In a recent paper in Development Genes Evolution., Caroti et al. (2015) reported on their efforts to extend the piggyBac transposon-based germline transformation system to the Diptera Megaselia abdita and Chironomus riparius and to use it for in vivo labelling of nuclei.

Transposon-based vectors still remain the most common tool for stable germline transformation in insects.

In Drosophila, it has facilitated in-depth genetic studies which otherwise otherwise would not have been possible, such as in vivo functional analysis of cis-regulatory DNA, gene knock-in and knock-out, generation of transgenic insects for applied purposes etc.

Chironomus riparius, egg mass, hatched eggs, first instar larvae and adult.

However, researchers working with non-drosophilid insects – especially in the field of evolutionary developmental genetics, where comparative analysis of variably related insects is required – are faced by the lack of protocols for stable germline transformation.

M. abdita and C. riparus are being used in studies of embryonic development in conjunction with Drosophila melanogaster.

Megaselia abdita

Caroti et al. (2015) used the standard piggyBac vector pBac{3xP3-eGFPafm} (Horn and Wimmer, 2000) to generate transgenic lines and demonstrated the feasibility of the TTAA piggyBac element for germline transformation of the two dipterans M. abdita and C. riparius.   They report an estimated germline transformation rate of 5.2% (2/38) and 5.5% (2/36) in M. abdita and 3.7% (1/27) in C. riparius.  The authors have also demonstrated the functionality of the artificial eye-specific promoter 3xP3 to drive the expression of eGFP in the eyes of adult M. abdita despite strong and dark pigmentation and in the nervous system of the last instar larvae of C. riparius as described by Horn et al. (2000).

http://dev.biologists.org/content/134/20/3571/F2.large.jpg

Typical DNA transposon and its conversion to a binary gene vector system

Furthermore, Caroti et al. explored the use of piggyBac vectors for in vivo labelling of nuclei. To do so, the authors have successfully generated two gateway versions of the popular plasmid pBac{3xP3-eGFPafm} and introduced thereby a fast, efficient, and ligation-free cloning method to generate piggyBac-based vectors for insect transgenesis: pBacDestA{3xP3-eGFP} and pBacDestB{3xP3-eGFP}. Using three way gateway reactions, the authors were able to simultaneously subclone three fragments (Histone2Av, mCherry, and spaghetti squash 3´UTR) to generate pBacDest{HistAv::mCherry}, which they used to establish M. abdita transgenic lines for studying the gastrulation process during early embryonic development in vivo, visually as well as computationally following individual nuclei.

Mechanism of piggyBac transposition.   Image credit Skipper et al. Journal of Biomedical Science 2013 20:92   doi:10.1186/1423-0127-20-92

Mechanism of piggyBac transposition. Image credit Skipper et al. Journal of Biomedical Science 2013 20:92 doi:10.1186/1423-0127-20-92

Each of these insects presents certain challenges with respect to delivering vectors and transposase and the authors’ description of their solutions could also be of some value to others looking for ideas as to how to inject embryos of species of interest to them.

Caroti, F., Urbansky, S., Wosch, M., Lemke, S., 2015 Germ line transformation and in vivo labeling of nuclei in Diptera: report on Megaselia abdita (Phoridae) and Chironomus riparius (Chironomidae). Dev Genes Evol 225: 179-186doi: 10.1007/s00427-015-0504-5.

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