TY  - GEN
T1  - Comparison of N- and C-terminally endogenously GFP-tagged WEE-1.3 strains in C. elegans
AU  - Fernando, Lourds M.
AU  - Golliday, Kyrionna
AU  - Boateng, Ruby
AU  - Allen, Anna K.
DO  - 10.17912/micropub.biology.000353
UR  - http://beta.micropublication.org/journals/biology/micropub-biology-000353/
AB  - The Wee1/Myt1 family of kinases are important cell cycle regulators during mitosis and meiosis, and act by specifically phosphorylating certain amino acid residues and inhibiting their target genes. The C. elegans WEE-1.3 protein is a member of this family and has been shown to play an essential role in maintaining oocyte meiotic arrest in the nematode (Allen et al., 2014; Burrows et al., 2006). A complete absence of WEE-1.3 results in embryonic lethality, while depletion of WEE-1.3 starting at the L4 larval stage results in precocious oocyte maturation and sterility (Allen et al., 2014; Burrows et al., 2006). Previously we have shown that WEE-1.3 is expressed throughout the soma and germ line of adult hermaphrodites and developing embryos via antibody staining, transgenic animals tagged with GFP at the N-terminus and C-terminus of WEE-1.3, and endogenously tagging the N-terminus of WEE-1.3 via CRISPR (Allen et al., 2014; Fernando et al., 2020). In these situations, the expression of WEE-1.3 is strongly perinuclear, but with cytoplasmic punctae that co-localizes with ER proteins in oocytes and embryos (Allen et al., 2014; Fernando et al., 2020). Here we have generated a homozygous wee-1.3::gfp strain (WDC8) where GFP is inserted at the 3′ end of the wee-1.3 genomic locus via CRISPR/Cas9 endogenous genome editing. The endogenous C-terminal WEE-1.3::GFP strain (WDC8) exhibits a perinuclear and cytoplasmic punctate localization expression pattern that is identical to that observed in the WEE-1.3 antibody stained germ lines, transgenic WEE-1.3::GFP animals and in endogenous gfp::wee-1.3 (WDC2) strains (Figure 1A-B). This expression is ubiquitous throughout the soma, in the germ line from the distal tip to the proximal oocytes, in developing embryos, and in sperm stored in the spermatheca. Importantly, both N- and C-terminally endogenously GFP tagged WEE-1.3 strains have a normal 24-hour brood size compared to wild-type control animals implying that we have not disrupted the function of this important reproductive protein kinase by fusing the GFP protein directly to it (Figure 1C). Our data suggest that the new C-terminally endogenously GFP tagged WEE-1.3 strain represents wild-type WEE-1.3 expression and activity, and can be used to monitor endogenous WEE-1.3 levels and localization appropriately.
PY  - 2021
JO  - microPublication Biology
ER  -