From rPERK to myc-tagged mPERK: Construction of pShuttle-loxp- tpA-loxp-mPERK_9E10-IRES- hrGFP-1 plasmid for conditional rescued PERK KO mouse.

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