From rPERK to myc-tagged mPERK:
Construction of pShuttle-loxp-tpA-loxp-mPERK_9E10-IRES-hrGFP-1 plasmid for conditional rescu
ed PERK KO mouse
EcoR V-rPERK- SmaILoxp-tpA-loxp
A myc-tagged mouse PERK became available
• Better to use mouse PERK in mouse system
• No good antibody available for PERK
Mysterious Plasmid Degradation
• Happened first time on pcDNA3.1+ extracted from transfected electro-competent cells– (Digested with EcoR I,
Nhe I and XhoI)
DNase Contamination?
• Extraction Kit?– No problem with other plasmids extracted
• Something Wrong with Enzymes?– Degradation happens even without enzymes
• Buffers contaminated?– Fine for other plasmids
• Problem disappeared after the previous kit was used up and the new one came in
Mysterious Plasmid Degradation Once More
• Ligation products of mPerk_9E10 and p-shuttle-loxp-tpa-loxp fragments extracted from transfected electro-competent cells get degraded in buffers.
Really Strong DNase
• Boiling of DNA for 5 min• Phenol-Chloroform Extraction and Alcohol repre
cipitation• Traditional alkaline lyses extraction
None of them worked
• Degrades other good plasmids when incubated together in enzyme buffer
• Strain problem? Transfect them into DMSO com
petent cells from JW.
Digest with Hind III
• Expected fragments : 8690, 2608, 1127, 894, 220
• Control (psh-tpa-rPERK) 8960, 3249, 1385
Future Work
• Transfect into regular cell line and TE-CRE cell line to confirm CRE-controlled mPERK_9E10 expression– DNA level: PCR detection of shortened version of pla
smid– RNA level: RT-PCR to detect myc-tag expression– Protein level: western-blot with myc-tag antibody– Functional level: More eIF2alpha phosphorylation upo
n ER stress