Southern blot analysis of the transcript produced from the CD43 promoter linked to luciferase and integrated within the K562 genome.

The mixed pool of K562 cells containing within their genome theCD43/luciferase fusion gene was treated for 24 hours with PMA and then total RNA isolated using an RNeasy Maxi Kit (Qiagen, Valencia, CA). A GeneRacer Kit (Invitrogen Life Technologies) was used to ligate the GeneRacer RNA Oligo specifically to the 5′ end of full-length mRNA, which was then reverse-transcribed using the GeneRacer Oligo dT Primer. The resulting products were used as templates in a PCR containing the primer LUC-4, which hybridizes to the coding strand of the luciferase gene, and the GeneRacer 5′ Primer, which represents the DNA equivalent of the 5′ end of the GeneRacer RNA Oligo. PCR products were then subjected to Southern blot analysis using as probe the radiolabeled oligonucleotide LUC-2, which hybridizes to the coding strand of the luciferasegene further 5′ than does LUC-4. The hybridization signal detected by autoradiography is depicted.