Prime a gene labeling system

To validate PCR-positive cell lines, genomic DNA was isolated (Gentra, Qiagen) from fibroblasts grown on the propagation culture dishes. Two to 10 ng of genomic DNA was whole-genome amplified (Repli-G, Qiagen) and digested with AflII and SspI. Following gel electrophoresis, samples were transferred to a positively charged nylon membrane (Roche Diagnostics) by using an alkaline transfer procedure. The membrane was briefly rinsed in 5× saline-sodium citrate (SSC) buffer, completely dried and subjected to UV crosslinking. The DNA probes for CLN3 and the NeoR cassette were produced by PCR amplification using primers pCLN3probeF2/pCLN3probeR3 and NeoR-F/NeoR-R, respectively (Table S2). Probes were radiolabeled with [α-³²P]dNTP by random priming using the Prime-a-Gene Labeling System (Promega), and the radioactive probes were purified using CHROMA SPIN+TE-100 columns (Clontech). Membranes were prehybridized in Rapid-hyb Buffer (Cytiva Amersham) for 30 min at 65°C; then, 25 μl of α-³²P-labeled probe was added and hybridization proceeded at 65°C for 2 h. The membrane was washed once in 2×SSC and 0.1% SDS at room temperature for 20 min and three times in 0.1× SSC and 0.1% SDS at 65°C for 15 min each. To confirm the animal genotype, high-molecular weight genomic DNA was isolated from miniswine umbilici. The remaining steps were performed as described above.

Genomic DNA was extracted from leaf tissue of greenhouse grown plants as described [16 (link)]. 15ug samples from individual plants were digested with EcoR1 and eletrophoresed on a 1% agarose gel at 20 V for 20 hr, transferred to a charged nylon membrane (Amersham/GE Healthcare, Baie d’Urfe, QC). After 3 hr of blocking at 55°C with hybridization buffer plus blocking reagent (Amersham) and NaCl 0.5 M, a probe encompassing the wtRfo promoter and coding sequences was labeled with α-³²PdCTP using the Prime-a-Gene Labeling system (Promega, Madison WI, USA). Hybridization was performed at 65°C overnight and the membrane was washed with 0.5% SSC and 0.5SDS% (W/V) 3 times for 5 minutes each at room temperature. The membrane was then washed twice for 30 minutes at 65°C and exposed to a Phosphor screen for 48 h at room temperature. The screen image was visualized using a PhosphorImager scanner (Amersham).

To verify integration of the hygromycin resistance cassette into the nuclear genome of P. indica, Southern blot analysis was performed. Genomic DNA from 7-day-old cultures grown on CM medium was extracted; 10–20 μg of extracted DNA was digested overnight with SacI (NEB). The digested DNA was separated on 0.9% TAE agarose gel for 5 h at 80 V and blotted onto a nylon membrane (AmershamBiosciences Hybond-N+, GE Healthcare) over night. The DNA was UV cross-linked to the membrane in a GS GENE LINKER UV chamber (BIO-RAD) using an auto cross-linking program (C2, 50 mμ Joule). The labeling of the Hygromycin B probe was performed using the Prime-a-Gene^® Labeling System according to the manufacturer’s instructions (Promega). Hybridization and washing steps were performed at 65°C. The membrane was exposed on phosphorimaging screens (Bio-Rad) and signals were detected using a molecular imager and the Quantity One software (Bio-Rad).