TY  - GEN
T1  - Triple and quadruple mutation of RGD motif using CRISPR-Cas9 in him-4 locus of Caenorhabditis elegans
AU  - Park, Aileen
AU  - Qiu, Zhongqiang
AU  - Lee, Myeongwoo
AU  - Lewis, Kendall
AU  - Cross, Margaret
AU  - Baur, Olivia
AU  - Brice, Sophia
AU  - Whiteley, Lauren
DO  - 10.17912/micropub.biology.000249
UR  - http://beta.micropublication.org/journals/biology/micropub-biology-000249/
AB  - The him-4 gene in Caenorhabditis elegans is involved in important developmental processes in the organism including basement membrane attachment, anchor cell invasion, defective cytokinesis, and gonad positioning along the basement membrane (Morrissey et al., 2014). The human ortholog to him-4, HMCN 1 (hemicentin-1), is implicated in age related macular degeneration 1 (Thompson et al., 2007). HIM-4/HMCN1 is a highly conserved extracellular matrix structural component and contains six RGD (Arg-Gly-Asp) cell-binding motifs within its 48 immunoglobin (Ig)-like repeats, while HMCN1 contains only one RGD motif (Vogel and Hedgecock, 2001). Two novel mutant alleles of C. elegans him-4 were generated using CRISPR-Cas9 gene editing in this study. In one mutant allele, three of the aforementioned six RGD motifs were mutated to RGE (Arg-Gly-Glu) (Takahashi et al., 2007). Four of the six RGD motifs were altered to RGE in the second mutant line. Neither of these mutant lines exhibited outstanding abnormal phenotypes when screened for behavioral or morphological abnormalities. We examined the mutants for Him (high incidence in male) phenotype, kq8207 (0% male, n=888) and kq8297 (0.1% male, n=963). However, the examined animals failed to show an outstanding number of males compared to N2 (0%, n=582). Mutants were also examined for behavioral abnormalities in a thrashing assay. A Mann-Whitney U Test revealed possible differences between kq8207 mutants and N2 worms (p=0.037), and revealed no significant difference between kq8297 mutants and N2 worms (p=0.447). 53 triple RGD mutants (average 19.85 thrashes), 51 RGD quadruple mutants (average 21 thrashes), and 51 N2 worms (average 22.16 thrashes) were used in this assay. Morrissey et al. demonstrated that PAT-3/INA-1integrin is essential for assembly of HIM-4/hemicentin puncta during anchor cell invasion (Morrissey et al., 2014). However, integrin and HIM-4 failed to colocalize in other tissues (Vogel and Hedgecock 2001; Morrissey et al., 2014). Although anchor cell invasion was not examined in this study, these him-4 RGD mutants hold potential for further research pertaining to the functions of cell to extracellular matrix binding domains in C. elegans.
PY  - 2020
JO  - microPublication Biology
ER  -