Mos1-mediated transgenesis to probe consequences of single gene mutations in variation-rich isolates of Caenorhabditis elegans

Abstract

Caenorhabditis elegans, especially the N2 isolate, is an invaluable biological model system. Numerous additional natural C. elegans isolates have been shown to have unexpected genotypic and phenotypic variations which has encouraged researchers to use next generation sequencing methodology to develop a more complete picture of genotypic variations among the isolates. To understand the phenotypic effects of a genomic variation (GV) on a single gene, in a variation-rich genetic background, one should analyze that particular GV in a well understood genetic background. In C. elegans, the analysis is usually done in N2, which requires extensive crossing to bring in the GV. This can be a very time consuming procedure thus it is important to establish a fast and efficient approach to test the effect of GVs from different isolates in N2. Here we use a Mos1-mediated single-copy insertion (MosSCI) method for phenotypic assessments of GVs from the variation-rich Hawaiian strain CB4856 in N2. Specifically, we investigate effects of variations identified in the CB4856 strain on tac-1 which is an essential gene that is necessary for mitotic spindle elongation and pronuclear migration. We show the usefulness of the MosSCI method by using EU1004 tac-1(or402) as a control. or402 is a temperature sensitive lethal allele within a well-conserved TACC domain (transforming acidic coiled-coil) that results in a leucine to phenylalanine change at amino acid 229. CB4856 contains a variation that affects the second exon of tac-1 causing a cysteine to tryptophan change at amino acid 94 also within the TACC domain. Using the MosSCI method, we analyze tac-1 from CB4856 in the N2 background and demonstrate that the C94W change, albeit significant, does not cause any obvious decrease in viability. This MosSCI method has proven to be a rapid and efficient way to analyze GVs.

Figure 1. The C94W change is within the TACC domain.

(a) Schematic representation of TAC-1 (1 to 260 amino-acid sequence). The majority of the protein is composed of the TACC domain which is depicted by the cyan box. Location and nature of all of the point mutants identified to date are shown as well. The ok3305 knockout allele, which removes the majority of tac-1 is depicted using a pink box. (b) The multiple sequence alignment of TAC-1 was adopted from Bellanger et al. 2007 . The positions of the previously isolated point mutants tac-1(or455) and tac-1(or369/402) are depicted using red stars, while the C94W change identified in CB4856 is depicted using a red arrow. The cyan box highlights the presence of the TACC domain. All of the known point mutations occur within the TACC domain, but none of them affect conserved amino acids.

Figure 2. A single-copy transgene insertion used to investigate consequences of single gene mutations in variation-rich isolates of C. elegans.

(a) Analysis of tac-1(or402) using Mos1-mediated transgenesis. tac-1, including its 5′ and 3′ regulatory sequences, was amplified using high-fidelity DNA polymerase from EU1004 genomic DNA and cloned into a pCFJ178 vector. The red dotted line located in the third exon of tac-1 depicts the or369/402 A to G change that results in an L229F amino acid change. Once cloned into the pCFJ178 vector, the transgene was inserted into the cxTi10882 Mos1 (depicted in orange) integration site on chromosome IV (depicted in purple). The resulting JNC152 strain contains both the endogenous copy of tac-1 located on chromosome II (depicted in blue), and tac-1 isolated from EU1004 inserted on chromosome IV, dotSi121. To uncover the effect of tac-1(or402), dotSi121 was examined in the absence of endogenous TAC-1 using ok3305. Thus, we constructed JNC153. (b) Schematic representation of the method used to investigate consequences of tac-1 variations detected in CB4856 tac-1, including the 5′ and 3′ regulatory sequences, was amplified using high-fidelity DNA polymerase from CB4856 genomic DNA and cloned into a pCFJ178 vector. The red dotted lines represent single nucleotide changes detected in CB4856 tac-1. Then, the transgene was inserted into the cxTi10882 Mos1 (depicted in orange) integration site on chromosome IV (depicted in purple). JNC150 contains both the endogenous copy of tac-1 located on chromosome II (depicted in blue) and tac-1 isolated from CB4856 dotSi120 inserted on chromosome IV. Then, dotSi120 was analyzed in the absence of endogenous tac-1(ok3305). (c) PCR bands of the expected size (6kb) for stably integrated single copy insertions of tac-1, dotSi121 and dotSi120.