Eventually, some clones were identified as heterozygous for mouse Pou3f2 and xPou3f2 (or Pou3f2 ΔGQP) with neo r by Southern blot screening using a 5′-probe, 3′-probe, and neo probe.
Blots were initially screened using the anti-phospho eNOS as per supplier's instructions (Cell Signalling Technology) and, following stripping, were subsequently re-screened using the anti-eNOS (Transduction Laboratories).
DNA extracted from the remaining lines were screened using the probe-based screen in conjunction with the TaPFT1 CAPS screen and in this case, CAPS gels were also blotted and hybridised to a TaPFT1 probe to ensure specificity.
To confirm that these repetitive sequences were present in a high copy number in the nurse shark genome and to test for their existence in related animals, we did Southern blotting and library screening using 'overgo' probes of NSRE1 and NSRE2.
To identify BAC clones containing ULBP genes, filter membranes containing the RPCI-42 male Holstein BAC library (12X genome coverage; Children's Hospital Oakland Research Institute) were screened by Southern blot hybridization using the full-length ULBP1 [GenBank: AF317556] cDNA clone as a probe.
The construction of the libraries, clone sequencing, vector and adaptor trimming and differential screening using dot blot hybridization were outsourced to Rx. Bioscience Ldt (Maryland, USA).
Pharmacological screening using other methods such as western blot can further confirm the relative pharmacological activity of the extracts.
Progeny were screened for transgene integration by slot blotting, using the human CD2 sequence as a probe, and by PCR.
