Pbluescript ks vector

RNA in situ hybridization was carried out as described using digoxigenin-labeled antisense riboprobes (Sciavolino et al., 1997 (link)). The full-length open reading frame (ORF) of Ldb1 was used to prepare the hybridization probe, which was amplified by PCR from retinal cDNA and subcloned into the pBluescript KS(-) vector (Agilent Technologies). EdU (5-ethynyl-2′-deoxyuridine) labeling was performed as previously described (Luo et al., 2012 (link)) using the Click-iT EdU labeling kit (Invitrogen).

Infectious clones corresponding to DNA-A and DNA-B of the viral isolates ToRMV-[BR:Ub1:96] [24 (link)] and ToSRV-[BR:PG1:Pep:03] [25 (link)] were used. These are the same clones used in the study of Silva et al. [23 (link)].
For confirmation of the sequences of the infectious clones, complete genome units were obtained by excision with BamHI (ToSRV DNA-A), EcoRI (ToRMV DNA-A) and KpnI (DNA-B from both viruses). After purification, these fragments were ligated into the pBLUESCRIPT-KS+ vector (Agilent, Santa Clara, CA, USA), previously linearized with the same enzymes. Recombinant plasmids were used for transformation into Escherichia coli by electroporation, and three clones of each genomic component were sent for commercial sequencing by primer walking (Macrogen Inc., Seoul, Republic of Korea). Sequences were assembled using DNA Baser v. 3.5 (Heracle Biosoft, Mioveni, Romania) and organized to begin at the nicking site in the invariant nonanucleotide at the origin of replication (5′-TAATATT//AC-3′). Pairwise sequence comparisons of DNA-A and DNA-B, CRs and ORFs between ToRMV and ToSRV were performed using Sequence Demarcation Tool (SDT) v.1.2 [26 (link)] with the MUSCLE alignment option [27 (link)].