Synthesis of PTEN mRNA via in vitro Transcription

Vector carrying open-reading frame (ORF) of PTEN was a gift from William Sellers^{74 (link)} (pSG5L HA PTEN wt; Addgene#10750). The vector was linearized by ApaI/EcoRI digestion and purified. HA-PTEN ORF under the regulation of T7 promoter was then amplified by PCR reaction. The amplicons were further purified and used as templates for in vitro transcription (IVT). The modified PTEN-mRNA was synthesized as described previously^{48 (link), 49 (link)}. In brief, IVT was conducted using MEGAscript T7 kit (Ambion) with 1–2 μg template and 7.5 mM ATP, 1.5 mM GTP, 7.5 mM 5-methyl-CTP, 7.5 mM pseudo-UTP (TriLink Biotechnologies), and 6 mM 3′−0-Me-m⁷G(5′)ppp(5′)G (anti-reverse cap analog, ARCA) (TriLink Biotechnologies). Reactions were incubated at 37°C for 4 hours, followed by Turbo DNase treatment for 15 min. 3´ poly(A)-tails were further added to IVT RNA products using a poly(A) tailing kit (Ambion). mRNA was purified by using the MEGAclear kit (Ambion), then treated with Antarctic Phosphatase (New England Biolab) at 37°C for 30 min, and further purified. Large-scale PTEN mRNA was custom-prepared by TriLink Biotechnologies as above (ARCA capped and enzymatically polyadenylated; fully substituted with Pseudo-U and 5’-Methyl-C; DNase and phosphatase treatment; Silica membrane purification) using 100–150 μg template containing T7 promoter and HA-PTEN ORF.