For luciferase or RFP reporters, the 3′UTRs of rab10 (ENSDARG00000046106) or prdm12b (ENSDARG00000007430) were cloned and fused to the ORF of luciferase and RFP in the psiCHECK2 and pCS2 plus (pCS2+) vectors, respectively.
For universal rab10 overexpression, full ORF and 3′UTR of rab10 was inserted to pCS2+ vector. To specifically overexpress rab10 in PGCs, the ORF of rab10 and gfp as well as the 3′UTR of nanos3 were inserted to pCS2+ vector to generate the pCS2-rab10-gfp-nos 3′utr plasmid.
All the point mutants including miR-202-5p binding sites within the 3′UTRs of rab10 or prdm12b, and the inactive or active rab10 mutants were constructed with hieff muttm multi-site-directed mutagenesis kit (Yeasen, Shanghai, China) as previously described [15 (link),45 (link)].
For mRNA synthesis, recombinant pCS2+ plasmids were linearized with NotI and transcribed using SP6 mMESSAGE mMACHINE Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.
For universal rab10 overexpression, full ORF and 3′UTR of rab10 was inserted to pCS2+ vector. To specifically overexpress rab10 in PGCs, the ORF of rab10 and gfp as well as the 3′UTR of nanos3 were inserted to pCS2+ vector to generate the pCS2-rab10-gfp-nos 3′utr plasmid.
All the point mutants including miR-202-5p binding sites within the 3′UTRs of rab10 or prdm12b, and the inactive or active rab10 mutants were constructed with hieff muttm multi-site-directed mutagenesis kit (Yeasen, Shanghai, China) as previously described [15 (link),45 (link)].
For mRNA synthesis, recombinant pCS2+ plasmids were linearized with NotI and transcribed using SP6 mMESSAGE mMACHINE Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.