Pmko 1 puro p53 shrna2

A SalI fragment of a human P53-knockdown construct (pMKO.1 puro p53 shRNA2, Addgene) was cloned into the SalI site of pinsB3 [25 (link)], resulting in pinsB3hP53sh. Finally, an AscI-SpeI fragment of pinsB3hP53sh was inserted into the AscI-NheI site of pPAC-2CAG-O2 (carrying two copies of CAG-driven Klf4, c-Myc, Sox2, and four copies of CAG-driven Oct4), resulting in pPAC-2CAG-O2hP53sh.
The reprogramming cassettes were introduced into 21HAC2 vectors [28 (link)] using the Cre-loxP system. Cre-recombinase expression vectors (pBS185; Invitrogen) (1 μg) and pPAC-2CAG-O2hP53sh (8 μg) were co-transfected into CHO/21HAC2, which are Hprt-deficient CHO (hprt^-/-) cells carrying a 21HAC2, in a 60-mm dish using Lipofectamine 2000. Recombinant clones were selected using HAT (Sigma) and 8 μg/ml Bsd two days after transfection. After 2 weeks, drug-resistant colonies with a functional HPRT allele were identified by genomic PCR and isolated. Next, quantitative RT-PCR analysis of reprogramming factors was performed. A CHO cell donor clone that stably expressed reprogramming factors comparable to CHO/iHAC2 was selected and designated CHO/iHAC/X53. FISH analysis showed that the iHAC vector was maintained independently from the host chromosomes in CHO/iHAC/X53.

OCI-AML3 cells [15 (link)] (human leukemia cell line kindly provided by Dr. Mark Minden, Ontario Cancer Center, Canada) and OCI-AML–3 cells stably expressing shRNA targeting p53 and vector control [16 (link)] (kind gifts from Dr. Paul Corn, University of Texas MD Anderson Cancer Center), mouse embryonic fibroblasts (MEF) ^wt/wt, ^{AMPK -/-} [17 (link)] (kind gifts from Dr. Juan Fueyo-Margareto, Neuro-oncology, MD Anderson Cancer Center) were cultured in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum (FCS). HL60, HEK-293T and REH cells were obtained from the ATCC (Manassas, VA). Phoenix Amphotrophic retrovirus packaging cells were obtained from Orbigen (San Diego, CA). A set of 7 shRNAmirs each targeting BECLIN 1 plus non-silencing control lentiviral vector were obtained from Open Biosystems (Huntsville, AL). Lentiviral packaging plasmids MD2.G (plasmid 12259) and psPAX2 (plasmid 12260), both constructed by the laboratory of Didier Trono, plus retroviral vectors pMKO.1 puro p53 shRNA#2 (plasmid 10672), pMKO.1 puro GFPshRNA (plasmid 10675), and pBABE-puro mCherry-EGFP-LC3B (plasmid 22418) (constructed in the laboratory of Jayanta Debnath) were obtained from Addgene (Cambridge, MA).