Modification of the human CD34 transgene by homologous recombination in bacteria to allow expression of the tetracycline transactivator (tTa).

The original PAC clone 88L2 (A) was modified to allow for the expression of the transactivator protein in tissues targeted by regulatory elements of the human CD34 locus. Shown is the transcription start site (horizontal arrow) and exon 1 (“ex 1”) with the location of the ATG translational start. The white box indicates the 5′ untranslated region, and the black box indicates the 27 amino acids of the human CD34 protein encoded by exon 1. Also shown are flankingBamHI, NotI, and HindIII sites located 1075 bp upstream and 360 bp and 2.7 kb downstream, respectively, from the transcription start site. The 1026-bp tTAgene26 was inserted directly at the promoter of the humanCD34 gene, such that the first ATG codon of theCD34 gene became the first codon of tTA, using the recombination cassette shown in panel B. An IRES element inserted downstream of tTA was followed by the sequence for humanCD34. A fragment containing FRT recombinase sites27 flanking the zeocin antibiotic resistance gene was inserted into human CD34 intron 1 and served as a selectable marker for homologous recombination. FRT recombinase was then used to delete the zeocin gene in the final transgene, shown in panel C.