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238 Cheng and Chang3.4.4. Fixation of Transfected Cells for Immunostaining1. Remove growth medium and wash the transfected cells three times with PBS.2. Fix the transfected cells with 3% paraformaldehyde for 1 min and wash the fixedcells three times with PBS.3. Permeabilize the fixed cells with 0.5% Triton X-100 for 15 min and wash the permeabilizedcells three times with PBS.4. Perform immunostaining with specific antibody according to standard protocols.4. Notes1. This approach is cost-effective and convenient, as any annealed oligonucleotideduplexes can be directly cloned into these four different expression vectors at thesame time.2. The main advantage of this approach is that the preparation of the inserting vectorsis simple and efficient by only double digestion with restriction enzymes ClaIand HindIII to remove the stuffer of Puro R gene from pHsH1puro, pHsU6puro,pMmH1puro, and pMmU6puro vectors. This will dramatically increase thecloning efficiency to more than 75%.3. The oligonucleotides used for constructing the systems can be purchasedfrom any local commercial suppliers without any further modification ortreatment.4. The sequential digestion of pGEM-7ZF(+) by EcoRI and HindIII followed byagarose gel purification is strongly recommended to ensure a complete digestionof vector by both restriction enzymes. This will greatly reduce the self-ligation ofvector in the cloning.5. The length of duplex region for a shRNA is relatively flexible from 19- to 29-nt.Although increasing the length of duplex region for a relatively ineffective 19-ntshRNA can increase its effectiveness, increasing the length of an effective 19-ntshRNA may not further improve the inhibition effect.6. The annealing of two complementary oligonucleotides can be efficiently carriedout in 1X T4 DNA ligase buffer, which can be obtained from any T4 DNA ligasecommercial suppliers.7. The annealed oligonucleotide duplexes do not need to be phosphorylated beforethe ligation step because it might result in multiple copies of insertion.8. It is important to construct the second expression cassette with the same orientationas both the human and mouse H1 promoters, because these two promoters couldexpress the protein-coding genes by the activity of Pol II-dependent poly(ADP-ribose)polymerase-2 promoter, efficiently. Otherwise the Pol II-dependent poly(ADP-ribose)polymerase-2 promoter could possibly transcribe the antisense strand of proteincodinggenes, resulting in formation of the long dsRNAs that might trigger nonselectivecytotoxic effects (44).9. By using this protocol for cloning the shRNA-expression cassettes, it is efficientand cost-effective that only four colonies are selected and screened for the positiveclones containing the shRNA-expression sequence.