Methods


Methods

In situ Hybridization
The gigantic polytene chromosomes of Sciara undergo twelve rounds of endoduplication in the larval salivary glands and therefore are even larger (8192C) than Drosophila (1024C) salivary gland polytene chromosomes. Due to their large size, Sciara polytene chromosomes provided the first chromosomes for development of the method of in situ hybridization in the lab of Joe Gall (Pardue et al 1970). Translocation break points within the rDNA tandem array were used to map the controlling element (Crouse 1960b) that controls X dyad non-disjunction and subsequent X chromosome elimination (Crouse et al. 1977; Crouse 1979).

Somatic cells contain three autosomes (chromosome II, III and IV) and the X chromosome that folds back upon itself to form a circle. Sciara polytene chromosomes do not form a chromocenter. The germ line contains a variable number (usually two) of germ line limited (L) chromosomes.

Tissue Culture

A culture medium has been devised (Cannon 1964) for maintenance of Sciara salivary glands, and it supports the normal developmental progression of DNA puffing (Cannon 1965). Cultured diploid cell lines from Sciara have not been developed.

Transformation

Senior research associate Yutaka Yamamoto in the Gerbi lab has successfully developed transformation methodology for Sciara. This required development of techniques for egg collection, embryo injection and selection of transformants. Yutaka has used a piggyBac vector to insert a cassette with target sites for a zinc finger nuclease, phiC31 attP, FRT, and loxP into the Sciara genome; this construct also contains selectable markers for Blasticidin resistance and ECFP driven by the highly conserved constitutive promoter containing three eyeless binding sites (3XP3) to identify positive transformants by visualizing ECFP fluorescence of the nervous system through the larval cuticle (Fig. 3). He used the Autographa carifornica nuclear polyhedrosis baculovirus enhancer-promoter (hr5-ie1) instead of the hsp70 promoter to drive piggyBac transposase; this dramatically increases its expression ~500 to 1000-fold. As a further improvement, Yutaka injected Sciara embryos with a hyperactive transposase (Wellcome Trust Sanger Institute) for more efficient integration of the piggyBac vector. In addition, he shortened the piggyBac vector to 2.7 kb, made possible in part by driving the Blasticidin resistance gene and ECFP from the same baculovirus promoter (hr5-ie1) and placement of the 2A sequence between these genes to cleave the polycistronic protein.

In addition, Yutaka has developed methodology for whole organism genome editing at specifically targeted sites in the Sciara genome, using targeted large DNA insertion via ObLiGaRe nonhomologous end-joining (NHEJ) in vivo capture (Yamamoto et al 2015). Targeted gene insertion of large pieces of DNA is a goal of genome editing and has been done in cultured cells but only in a handful of whole organisms. The existing method to integrate foreign DNA using the homologous recombination (HR) pathway is inherently low efficiency, and many systems are refractory to this method (Orlando et al. 2010; Cristea et al. 2013; Weinthal et al. 2013). Our method utilizes the preferred pathway of NHEJ via site-specific cleavage, end-capture of cohesive ends and obligate ligation-gated recombination (Maresca et al. 2013) as a high efficiency approach to insert large pieces of DNA into specific target sites. We have demonstrated the efficacy of this method by insertion of 6.5 kb into a desired target site in the Sciara genome (Yamamoto et al. 2015). Our methodological advance is broadly applicable to a wide range of biological systems.