Can TRC hairpins in the pLKO.1 vector be subcloned into one of the shRNAmir vectors (pSM2, pSMP, LMP, TMP, pGIPZ, or pTRIPZ)?
There is no protocol for subcloning a TRC hairpin from the pLKO. 1 vector into any of the shRNAmir vectors since the TRC and shRNAmir constructs were developed by two different institutions. We don't guarantee the results, but you can use the RNAi Central hairpin design tool on the Cold Spring Harbor Laboratories website (http: //cancan. cshl. edu/cgi-bin/Codex/Codex. cgi) to microRNA-adapt the target sequence of the TRC hairpin for cloning into the shRNAmir vectors. The Paddison et al. (2004) paper also provides (Paddison, P et al (2004) "Cloning of short hairpin RNAs for gene knockdown in mammalian cells" Nature Methods Vol1: 2, 163-167) guidelines on how to do this. Please note that although the reference is specific for cloning into the pSM2 vector, the same protocol would apply for cloning into the LMP, TMP, pSMP or pTRIPZ vectors. Please also note that microRNA-adapted hairpins cannot be directly cloned into the pGIPZ vector but can only be subcloned into the pGIPZ vector from pSM2, pSMP or pTRIPZ. ** Please note the TMP/LMP vectors have been discontinued