Figure 1.
ot900 is a novel null allele of ceh-14. We used gRNAs located in the first and last exons of ceh-14 to generate a large deletion allele spanning the ceh-14 ORF (A). The remaining 35bp in exon 1 are capable of generating an 11aa peptide before a deletion-induced frame-shift results in a premature stop (B).
Description
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.
Reagents
OH15422 ceh-14 (ot900) X. Will be available at CGC.
Acknowledgments
Funding
This work was supported by the HHMI and NIH (R37NS039996, O.H., F31NS096863, E.A.B.)
References
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