Figure 5.

The multigene structure of the hGH locus was critical for the appropriate activation of the placental hCS genes. (A) Replacement of the hGH gene cluster with a single PGR unit (LCR-CSA/BAC transgene). Replacing the hGH cluster with a single hCS-A PGR unit involved truncation of the 3′ terminus of the B-cell specific CD79b gene. Thus, any transcription initiated from the B-cell promoter of the CD79b gene in contaminating B cells would have the possibility of extending into the CS-A locus (‘mRNA1’). To specifically detect transcripts originating from the hCS promoter (‘mRNA2’ originating at the hCS promoter) we used the RT/PCR primer set shown in the diagram. (B) hCS-A was robustly expressed from the LCR-CSA/BAC transgene but lacked copy-number dependence. CoRT/PCR-TaqI analysis (as in Figure 2C) confirmed robust hCS-A mRNA expression from the LCR-CSA/BAC transgene (left panel). However the levels of the expression per transgene copy demonstrated a marked line-to-line variation (r² = 0.59) (right panels). (C) Expression of hCS from the LCR-CSA/BAC transgene demonstrated a dramatic loss of tissue specificity. Tissue survey of expression from the indicated hGH transgene loci was analyzed by RT/PCR.