Flies were reared on standard cornmeal medium at 25 ℃. The following strains were used: w1118, tubublin-Gal4, daughterless-Gal4,Cg-Gal4, ppl-Gal4,β2tubulin-GFP; Bloomington Drosophila Stock Center Df(3R)BSC478/TM6(BL24982), Dj-GFP (BL5417), Pif1A-GFP.FPTB/Cyo(BL42670), Dj-GFP(BL5417), protamine B-eGFP/Cyo(BL58406); bam-Gal4 and tj-Gal4 (kindly provided by Chao Tong); nos-Gal4 and UAS-actin.GFP/CyO; Sb/TM6B (Tsinghua Fly Center).
To generate the Pif1A mutants, we employed the “CRISPR/Cas9 method”, the gRNA was designed to recognize a 19-nt target sequence and act to direct Cas9-mediated cleavage of both DNA strands within the target site.
To generate UAS-Pif1A-RI, UAS-Pif1A-RG, and UAS-eGFP-Pif1A-RG transgenenic flies, we amplified the full length Pif1A cDNA with the primers below and cloned it into the pUAST-attb vector. These constructs were then transformed into VK33 embryos using the standard P-element-mediated transgenesis protocol.
Pif1A- RG-FCTGCGGCCGCGGCTCGAGATGGCTGAAAACCAAACCAAAACG
Pif1A- RG-RTCACAAAGATCCTCTAGATCAGAGCCGGGCATTCTCGGACGG
Pif1A- RI-FCTGCGGCCGCGGCTCGAGATGGGCAACGAGGAATCCT
Pif1A- RI-RTCACAAAGATCCTCTAGACTATTTCTTAGCTCTGAACAAG
eGFP-Pif1A- RG-F TTCGTTAACAGATCTGCATGGTGAGCAAGGGCGAGGA
eGFP-Pif1A- RG-R ATCTCGAGCCGCGGCCGCCACTTGTACAGCTCGTCCATG
Yuan X., Zheng H., Su Y., Guo P., Zhang X., Zhao Q., Ge W., Li C., Xi Y, & Yang X. (2019). Drosophila Pif1A is essential for spermatogenesis and is the homolog of human CCDC157, a gene associated with idiopathic NOA. Cell Death & Disease, 10(2), 125.
+ Open protocol