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. 2014 Feb 21;42(8):4906–4921. doi: 10.1093/nar/gku139

Figure 2.

Figure 2.

The hGH transgene locus is appropriately configured and expressed in the mouse placenta. (A) DNase I HS mapping of placental chromatin from transgenic mouse embryos carrying the intact hGH/BAC. Chromatin samples were exposed to DNase I for the times indicated to generate partial digestion products. The DNAs were then extracted, digested with EcoRI and analyzed by indirect-end labeling Southern blotting, as indicated in the diagram below the autoradiograph. The intact 23-kb EcoRI fragment encompassing the HSIII–V region is indicated by the gray arrowhead to the left of the autoradiograph and the sub-fragments generated by DNase I cleavage at HSIII, IV and V are indicated by black arrows and the black dots. A corresponding analysis of chromatin from a human placental choriocarcinoma line, JEG3, served as a positive control for placental HS formation. The pattern seen is consistent with that generated from normal human term placenta (4). Notably, two sub-bands (indicated by white dots) were observed in the Southern blot. These bands were detected consistently in the human placental chromatin and were previously demonstrated as non-specific bands unrelated to DNase I treatment (4,5). (B) Expression of the intact hGH/BAC transgene demonstrated appropriate tissue specificity. RT-PCR analyses of mRNAs isolated from the indicated tissues are shown. The primer set co-amplifies mRNAs generated from all five genes in the hGH cluster. GAPDH mRNA served as loading control. (C) Expression pattern from the intact hGH/BAC transgene in the mouse placenta recapitulated the expression from the native hGH locus in the human placenta. A 32P-labeled cDNA generated from co-amplification of all five of the hGH/hCS mRNAs (as in B above) was digested by TaqI to distinguish the expression of the individual mRNA species (see Materials and methods section). The products revealed robust expression of hCS-A/hCS-B mRNAs, much lower expression of hGH-V mRNA, and only trace detection of the hCS-L pseudogene transcript and the pituitary-specific hGH-N mRNA. This pattern recapitulated that observed in the human term placenta (5).