Pcr2.1 topo vector backbone

The wild-type (WT) lin-4 (+) construct (pHZ018) consists of a 695-nucleotide (nt) genomic fragment, which rescues lin-4 (e912) phenotype, in a pCR2.1-TOPO vector backbone (Invitrogen). This genomic fragment contains the 94-nt lin-4 precursor sequence and flanking genomic 5′ (498 nt) and 3′ (103 nt) sequences. Different lin4_m constructs were made using mutation-containing primers to amplify pHZ018 and replacing WT lin-4 in pHZ018 with the amplified PCR product. The specific mutations made for each construct are illustrated in Figure 1C. The lin4_m constructs (30 ng/µL or 17 ng/µL) were individually injected into PD7143 [lin-4 (e912)/mIn1 II; pha-1(e2123ts) III; rde-1(ne300) V] with a selection marker, pC1 [pha-1(+), 120 ng/µL or 133 ng/µL, respectively] (Granato et al. 1994 (link)). Transgenic lines derived from these injections were assayed for lin-4 activity [sublines without the pharyngeal mIs14(gfp) are homozygous for the chromosomal lin-4(e912) deletion mutation]. We note that transgenic strains with wild-type lin-4 are subject to differing degrees of partial rescue and mosaicism, with perfect rescue (full phenotypic rescue and egg-laying) observed in only a fraction of transgenic animals (Supplemental Fig. 2ai). Data for WT lin-4 rescue were taken from our previous work (Zhang et al. 2011 (link)).

The wild-type let-7 construct (CZ234, gift from Robin Trujillo and Chang-Zheng Chen) consists of a 2460-nt genomic fragment in a pCR2.1-TOPO vector backbone (Invitrogen). This genomic fragment contains the let-7 precursor sequence and flanking genomic 5′ (1747 nt) and 3′ (614 nt) sequences. Different let7_m constructs were made using mutation-containing primers to amplify CZ234 and replacing WT let-7 in CZ234 with the amplified PCR product. The specific mutations made for each construct are illustrated in Figure 2D. The let7_m constructs (m1-m13, 3–6 ng/µL) were individually injected into PD5584 [mnDp1(X;V)/+ V; let-7 (mn112) unc-3(e151) X] with pPD117.01 (pmec-7::gfp, 12–50 ng/µL) and CZ233 (10–44 ng/µL). Transgenic lines derived from these injections (uncoordinated animals with green fluorescent touch receptor neurons) were assayed for let-7 activity (rescue of vulval “bursting” phenotype). The let7_m constructs (m14-m15, 3–6 ng/µL) were individually injected into PD5585 [let-7 (n2853) X] with pPD117.01 (pmec-7::gfp, 12–50 ng/µL) and CZ233 (10–44 ng/µL). Transgenic lines derived from these injections (green fluorescent touch receptor neurons) were assayed for let-7 activity (rescue of vulval “bursting” phenotype) at 25°C.