Xl 10 gold e coli

All plasmid construction using polymerase chain reaction (PCR) was performed with PrimeSTAR Max DNA Polymerase (TaKaRa, Tokyo, Japan) on a PCR thermal cycler (Dice Touch, TaKaRa). Plasmids and PCR products were digested with appropriate restriction enzymes (New England BioLabs, Ipswich, MA, USA) at optimal conditions. Plasmid and PCR fragments were assembled with either NEBuilder HiFi DNA Assembly Master Mix (New England BioLabs, Ipswich, MA, USA) or DNA Ligation Kit Mighty Mix (TaKaRa). Chemically-competent Stbl3 E.coli (Thermo Fisher Scientific, Waltham, MA, USA) was transformed with AAV plasmids, and chemically-competent XL10-Gold E. coli (Agilent Technology, Santa Clara, CA, USA) was used for the other plasmids. The E. coli was grown in LB medium (Kanto chemical, Tokyo, Japan) on an LB plate containing ampicillin (100 μg/ml). Plasmids were inspected by diagnostic digestions with appropriate restriction enzymes and sequenced before virus production.

Lentiviral particles were created using the ViraSafe lentiviral packaging system (Cell Biolabs). ShRNA oligo sequences were based upon the Sigma-Aldrich MISSION shRNA library and were obtained from Integrated DNA technologies. The following shRNAs were used in this study: PCK1 sh3 (TRCN0000196706), PCK1 sh4 (TRCN0000199286), PCK1 sh5 (TRCN0000199573), shControl (SHC002), mouse PCK1 sh64 (TRCN0000025064), mouse PCK1 sh66 (TRCN0000025066), DHODH sh2 (TRCN0000221421), and DHODH sh3 (TRCN0000221422). Forward and reverse complement oligos were annealed, cloned into pLKO, and transformed into XL10-Gold E. coli (200314, Agilent). For PCK1 overexpression, PCK1 cDNA (plasmid ID HsCD00045535) was obtained from the PlasmID Repository at Harvard Medical School and cloned into pBabe-puromycin or plx304-blasticidin. For tetracycline-inducible experiments, the seed sequences of shRNA control (SHC002) or PCK1 sh4 (TRCN0000199286) were cloned into pLKO-Tet-On (Wiederschain et al., 2009 (link)). All plasmids were isolated using the plasmid plus midi kit (Qiagen). Transduction and transfection were performed as described previously (Yu et al., 2015 (link)).