An Accumulative Site-Specific Gene Integration System Using Cre
Recombinase-Mediated Cassette Exchange
Yujiro Kameyama, Yoshinori Kawabe, Akira Ito and Masamichi Kamihira
Department of Chemical Engineering, Faculty of Engineering, Kyushu University
Supplementary Figure Legends
Supplementary Figure 1.
Sequence analysis for loxPs associated with recombination
reactions in the plasmids (P1232Cmr and P12323Kmr) after the third and fourth
reactions. (Upper) The genetic sequences of loxP[wt] and the first loxP3 (upstream of
an IRES/DsRed cassette) in P1232Cmr were analyzed. The loxPs were sequenced using
Pr1 (for loxP[wt]) or Pr5 (for loxP3) as the sequencing primer. (Lower) The genetic
sequences of loxP[wt] and the first loxP6 in P12323Kmr were analyzed. The loxP[wt]
was sequenced using Pr1 as the sequencing primer. The first loxP6 was sequenced using
Pr6 as the sequencing primer for a DNA fragment generated from PstI-digested
P12323Kmr to distinguish from another loxP6.
Supplementary Figure 2.
Sequence analysis for loxPs associated with the
recombination reaction in the genome of CHO cells after the first and second reactions.
The genetic sequences of loxP sites residing on the genome of CHO cells were analyzed
using DNA fragments amplified from genomic DNA of CHO cell clones (CHO/P12 and
CHO/P123Neor) by PCR. (Upper) DNA fragments amplified from genomic DNA of
CHO/P12 cells using the Pr1 and Pr3 (for loxP[wt] and loxP4), or the Pr4 and Pr2 (for
loxP3) as the primers were applied for sequencing analysis of loxPs generated in the
genome of cells after the first RMCE reaction. (Lower) DNA fragments amplified from
genomic DNA of CHO/P123Neor cells using the Pr1 and Pr7
(5’-ACACCGGCCTTATTCCAA-3’) (for loxP[wt] and loxP1), or the Pr6 and Pr3 (for
loxP6) as the primers were applied for sequencing analysis of loxPs generated in the
genome of cells after the second RMCE reaction.