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. 2009 Feb 17;37(6):e44. doi: 10.1093/nar/gkp058

Figure 4.

Figure 4.

De novo construction of a human STAT3 mini-locus on 25-4 vector. (a) Strategy for modifying and inserting genomic DNA from BAC into chromosome vector. TT2F/25-4 cells that contained the human STAT3 genomic gene were selected with puromycin and drug-resistant cell lines were confirmed by PCR with CAG promoter and puro sequences, and by FISH using STAT3 and BAC sequences as probes. (b) The genomic composition of human STAT3 genes. Clone 2047G9 contains 139-kb of the upstream and 5-kb of the downstream regions of human STAT3. Clone 1781E2 contains 15-kb of upstream and 85-kb of downstream regions of human STAT3. The numbers in the boxes delineate exons. (c) FISH analysis of mouse ES cells containing human STAT3 mini-locus. Metaphase chromosomes were hybridized with human STAT3 cDNA (green) and BAC vector (red) sequences. Arrowhead shows 25-4 containing human STAT3; arrows show endogenous mouse Stat3. (d) Expression of human STAT3 from 25-4. The 2.2-kb fragments from human STAT3 transcripts were detected by RT–PCR in all ES cell lines that contained human STAT3. (e) Detection of human STAT3 products in TT2F/25-4 containing human STAT3. The STAT3 products were detected by anti-human STAT3 antibody in methanol-fixed cells. Green signals show human STAT3 products. (f) Expression of human STAT3 after long-term culturing. The TT2F/25-4 cells containing human STAT3 were cultured for 60 and 100 days and subsequently analyzed by RT–PCR. Human STAT3 transcripts were detected as before culturing.