Journal: Molecular Biology of the Cell
Article Title: Shortened nuclear matrix attachment regions are sufficient for replication and maintenance of episomes in mammalian cells
Figure Lengend Snippet: Analysis of state of existence of the transgene in CHO cells and copy number. (A) Rescue experiments in E. coli with Hirt extract from CHO cells transfected with MAR-3, MAR-4, and MAR-5. M: DNA marker; lanes 1, 3, 5: original plasmids with MAR-3, MAR-4, and MAR-5 double-digested with Kpn I and Bam HI, respectively; lanes 2, 4, 6: rescued plasmids with MAR-3, MAR-4, and MAR-5 double-digested with Kpn I and Bam HI 50 generations after transfection, respectively. (B) Southern analysis of DNA isolated from CHO cells transfected with MAR-3, MAR-4, and MAR-5. The hybridization pattern of one representative clone is shown for each construct. M: DNA marker; lane 1, 3, 5: original plasmids with MAR-3, MAR-4, and MAR-5 digested with Bam HI, respectively; lane 2, 4, 6: rescued plasmids with MAR-3, MAR-4, and MAR-5 digested with Bam HI 50 generations after transfection, respectively. Lane 7: Eco RI digestion of untransfected CHO cells; lanes 8, 9: CHO cells transfected with pEGFP-C (chromosomal DNA was isolated, digested with Eco RI, separated on a 0.8% agarose gel, blotted, and hybridized with pEGFP-C1 probe). (C) FISH analysis of eGFP served as a probe in CHO cells transfected with vector no eGFP gene (negative control), no MAR (pEGFP-C1), full-length MAR, MAR-3, MAR-4, and MAR-5, respectively. The vector without eGFP gene can only see blue metaphase chromosomes, integration vector pECFP-C1 can see vector insert into chomosomes, vector with full-length MAR, MAR-3, MAR-4 and MAR-5 was episomal state on metaphase chromosomes (blue: metaphase chromosomes; red: vectors). (D) The gene copies of each metaphase plate as determined by FISH analysis. Fifty metaphase spreads were analyzed by FISH for each clone, an average vector copy number was estimated, and SEM is indicated. (E) The copy number was assessed by qPCR analysis. A serial dilution with a plasmid containing the eGFP gene was used to determine the absolute copy number. Three independent experiments were performed in this study. SEM is indicated (Student’s t test, * P
Article Snippet: Bidirectional cloning was achieved by digesting the resulting PCR fragments with Kpn I and Bam HI restriction enzymes (Takara Biotechnology, Dalian, China) and inserting these sequences downstream of the eGFP expression cassette region in pEGFP-C1 ( ) to generate plasmids containing different subfragments of β-IFN MAR (MAR1–5). pEPI (i.e., full-length MAR) was used as a control ( ).
Techniques: Transfection, Marker, Isolation, Hybridization, Construct, Agarose Gel Electrophoresis, Fluorescence In Situ Hybridization, Plasmid Preparation, Negative Control, Real-time Polymerase Chain Reaction, Serial Dilution