A novel null allele of C. elegans gene ceh-14
Year: 2018
Type: article
Abstract: We have generated a novel null allele, ot900, of the C. elegans LIM homeodomain gene ceh-14. All existing deletion alleles of ceh-14 were generated using a Tc1 transposon insertion, one of which, ch3, is a putative null based on the resulting frame-shift and a reduction in detectable mRNA and protein levels (Cassata et al. 2000). However, in our hands we have noticed a propensity of the ch3 allele to revert to wild-type after several generations, perhaps due to a more complex rearrangement of the locus caused by the Tc1 transposon than previously appreciated. To avoid potential complexities of working with the ch3 allele, we used CRISPR/Cas9 to generate a new null allele of ceh-14. The ot900 allele was isolated with gRNAs targeted to the first and last exons of ceh-14 (gRNA #1: CTTGGCGAGTGCGATGAGC, gRNA #2: GTACTGTGGAGTCATGTGT; see Fig. 1), and then PCR screening for resulting large deletion alleles. Our primers for PCR genotyping of the deletion are 5’-CTCAACTAAATCGTCAGAATCGTC-3’ and 5’-GAGATGTATGAAACGAGCGAGCG-3’ which generate a 620bp product in the context of the ot900 deletion. The ceh-14 locus in the ot900 allele is only capable of generating an 11aa peptide due to the remaining bases in the first exon, before reaching a premature stop codon (Fig 1B). Previous studies have implicated ceh-14 in differentiation and function of the phasmid neurons, but have shown incomplete penetrance and variability in phenotypes resulting from the ceh-14 (ch3) allele (Kagoshima et al. 2013, Serrano-Saiz et al. 2015). We used the srg-13 marker of PHA identity (which was expressed in 20/20 wild-type animals) to assess whether phasmid neuron differentiation is more severely affected in ot900 than ch3, and found that this marker was indeed lost in 100% of animals (expressed in 0/15 animals), despite not finding an appreciable effect in ceh-14 (ch3) (expressed in 15/15 animals). This suggests that some of the variability in phenotype may be the result of the ch3 allele itself, rather than a property of CEH-14 function.
Source: PubMed Central
DOI: 10.17912/g434-3d85
Authors: Bayer, Emily A., Hobert, Oliver
Institutions: Columbia University, Howard Hughes Medical Institute
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2966793963