Construction of pEGFP-N1-MIC1 Plasmid and Its Effects on Mouse Forestomach Carcinoma Cells

Construction of pEGFP-N1-MIC1 Plasmid and Its Effects on Mouse Forestomach Carcinoma Cells

1. Materials

The following materials were used in this study:

• Cell line: Mouse Forestomach Carcinoma (MFC) cell line from Wuhan Procell Life Technology Co., Ltd.* Reagents: * GoTaq qPCR Master Mix (M722) from Promega * Endo-Free Plasmid DNA Midi Kit (D6915) from Omega * Restriction endonucleases QuickCut HindIII (1615) and QuickCut EcoRI (1605) from Takara Bio Inc. * pMD18T vector, pEGFP-N1 vector, 1 Kb DNA Ladder (3426A), DL1000 DNA Marker (3591A), and RNAiso Plus (9108) from Takara Bio Inc. * Anti-FLAG rabbit polyclonal antibody (RG001060) from Cell Signaling Technology * Beta-actin (20536-1-AP) antibody and HRP-conjugated Affinipure Goat Anti-Rabbit IgG (SA00001-2) from Proteintech * RPMI-1640 (01-100-1A) and fetal bovine serum (FBS) (04-001-1A) from BI, Kibbutz Beit Haemek, Israel * Lipofectamine TM 2000 from Invitrogen * Primers synthesized and sequencing conducted by Thermo Fisher.

2. Methods

2.1 Cell Culture and Transfection

MFC cells were cultured in RPMI-1640 supplemented with 10% FBS and antibiotics. Cells were transfected with the pEGFP-N1-MIC1 plasmid using Lipofectamine TM 2000.

2.2 MIC-1 Gene Amplification and Cloning

Total RNA was extracted from healthy mouse gastric tissue and reverse transcribed into cDNA. The MIC-1 gene was amplified by PCR using primers with HindIII and EcoRI restriction sites. The PCR product was then ligated into the pMD18T vector and transformed into E. coli DH5α.

2.3 Construction of pEGFP-N1-MIC1 Plasmid

The MIC-1 gene was excised from the pMD18T-MIC1 plasmid and ligated into the pEGFP-N1 vector. The resulting pEGFP-N1-MIC1 plasmid was confirmed by sequencing.

2.4 Western Blotting

Western blotting was performed to confirm MIC1 protein expression in transfected cells using an anti-FLAG antibody.

2.5 Cell Cycle Analysis

Cell cycle analysis was conducted using flow cytometry to assess the effect of MIC1 on cell cycle progression.

2.6 Cell Migration Assay

A scratch assay was used to evaluate the effect of MIC1 on cell migration.

2.7 Colony Formation Assay

Colony formation assay was performed to assess the effect of MIC1 on cell proliferation and colony formation ability.

3. Results

(This section would include the results obtained from the experiments described in the methods section).

4. Discussion

(This section would discuss the significance of the results and their implications for understanding the role of MIC1 in Mouse Forestomach Carcinoma).