Plvx lentiviral vector

Truncated 53BP1 (amino acids 1220–171120 (link)) was PCR amplified from 53BP1-YFP (kind gift from Prof. Galit Lahav, Harvard Medical School, Boston, MA). The PCR product was cloned into the pLVX lentiviral vector (632562, Clontech) containing a C-terminal Apple fluorescent protein (27698, Addgene), using In-Fusion cloning (639645, Clontech) and the BsrGI and XbaI restriction enzyme sites on the pLVX vector. The 53BP1_trunc-Apple insert was sequenced in its entirety.
The H2B-GFP reporter was created by replacing Apple fluorescent protein in pTag-H2B-Apple49 (link) (FP176, Evrogen) with pAcGFP1 (632492, Clontech). pTag-H2B-Apple was digested using the AgeI and NotI restriction enzymes, while pAcGFP1-HyG-C1 was digested using the AgeI and BspOMI restriction enzymes. pAcGFP1 was then ligated into the pTag-H2B vector and the resulting insert was sequenced in its entirety.

pLVX lentiviral vector was purchased from Clontech (USA). Polymerase chain reaction (PCR) was conducted using templates of IGHG1 specific shRNA and full length of IGHG1 cDNA sequences. PCR products were firstly purified using 1 % agarose gel and double digested by BamHI and EcoRI along with empty pLVX vectors. Ligation reaction was performed overnight between vector and the purified PCR products by T4DNA ligase. Ligation product was further used for the E.coli DH5α competent cells transformation. Cell clones were subsequently placed into the ampicillinum containing-LB plate and were incubated overnight at 37 °C. Positive clones were gathered and the plasmid was extracted and sequenced (Shanghai Invitrogen Biotech Co., Ltd). Afterwards, the lentiviral vectors carrying the IGHG1 shRNA / cDNA and control vectors were packaged and subsequently added into gastric cancer tumor cell groups with multiplicity of infection (MOI) of 20 (control group with empty vectors, overexpression group with IGHG1 cDNA vectors, suppressed group with IGHG1 shRNA vectors), and the vectors titer was set as 1 × 10⁹/mL. Then the fluorescent protein expression level was detected and transfection efficiency was evaluated 24–48 h post-transfection.

In order to distinguish NC from NP cells, NC were labelled with mEmerald fluorescent protein using lentiviral transduction. Lentiviral expression vector was generated by subcloning mEmerald coding sequence by PCR from pmEmerald-LifeAct-7 vector (Addgene, 54148) into pLVX lentiviral vector (Clontech, United States). For lentivirus production, Lenti-X 293T cells (Clontech, United States) were transfected with lentiviral expression vector and 3rd generation packaging plasmids prMDLg/pREE, pRSV-Rec, and pMD2.G (Addgene, 12251, 12253, and 12259, respectively) using Lipofectamine 2000 (Lifetech, 11668019). After 72 h, the supernatant containing lentiviral particles was collected, and lentiviral titer was assessed by ELISA using Quick Titer Lentivirus titer kit (Cell Biolabs, VPK-1070). Lentiviral transduction of NC was performed according to previously established protocol, reported affecting neither proliferation nor the differentiation capacity of chondrocytes (Miot et al., 2010 (link)). Briefly, NC were seeded in 6-well plates as 2.5 × 10⁶ cells/well and transduced with mEmerald-lentivirus at MOI 5 in the presence of 8 µg/ml polybrene, which yields ≥95% transduction efficiency (TEf). TEf was monitored by yielded the percentage of green-positive cells by flow cytometry (Aria III, BD) 3 days post-transduction.