Figure 4

Analysis of stable cell lines 0.5 μg of BACGFPNeoOE DNA was transfected into B16F10 cells with Lipofectamine 2000 and 6 clones were expanded in G418 selection. a) The percentage of cells expressing GFP was determined by FACs analysis at 30 days. B16F10 indicates untransfected cells. b) Analysis of DNA rescued from the 6 stable cell lines. DNA was extracted from the 6 cell lines and re-transfected into E. coli. DNA from 3 E. coli clones are shown for each B16F10 cell line as indicated above the lanes. Cell line number 2 gave rearranged BAC DNA in two E. coli clones, while the third is unrearranged. DNA was cut with SalI and resolved on a 0.3% SeaKem Gold agarose gel. Size marker M1 is the 1 kb DNA Ladder from Promega and M2 is the Lambda DNA-Mono Cut Mix from Biolabs. In between the two markers is the input BAC DNA cut with SalI. The sizes of the markers and BAC fragments are shown on the left and right of the gel respectively. c) Pulsed-field gel showing that the BAC DNA rescued from the cell lines is the same as the input DNA. The lanes carry; Low Range PFG marker (Biolabs)(M), input BACGFPNeoOE DNA cut with SalI (input), and DNA from three E. coli clones from cell line number 4 (4a, 4b and 4c) cut with SalI. The sizes of the markers and BAC fragments are indicated on the left and right of the gel respectively.