Schematic representation of the PCR strategies used to produce shRNA expression vectors. (A): The two-step PCR method used to generate pBovineU6-shEGFP. The 1st PCR amplified the bovine U6 promoter, EGFP shRNA sense, loop, and 3 nt of EGFP shRNA antisense using primers LL16 and LL23. The 2nd PCR amplified the bovine U6 promoter and the remaining EGFP shRNA components including EGFP shRNA antisense, terminator and XbaI using primers LL16 and LL13. (B): The one-step PCR method used to generate pMouseU6-shEGFP, pBovineU6-shScrambled, pMouseU6-shGapdh and pBovineU6-shGapdh. PCR reactions used forward primers paired with single reverse primers comprising all shRNA components. All final PCR products consisted of a mouse or bovine U6 promoter, shRNA sense, loop, shRNA antisense, termination sequence and XbaI site.