HSIII-HSV is insufficient to activate hCS-A expression in the transgenic placenta. (A) Transgene constructs. Each of the two transgene constructs is shown below the map of the hGH locus. Each of the four P-elements was named to correspond with its adjacent gene's name (Pl, Pa, Pv, and Pb), and the three enhancers were named similarly (El, Ea, and Eb). The hGH/BAC transgene encompasses the entire region. The HSIII-V/Pa(CSA)Ea transgene is composed of an 11.5-kb fragment encompassing HSV through HSIII ligated directly to a 7.5-kb hCS-A gene fragment that includes its contiguous 5′ and 3′ sequences, as shown. Sets of transgenic mouse lines were generated with each of these two constructs. (B) Expression of the hGH cluster genes in the transgenic placentas. Four lines of mice carrying the HSIII-V/Pa(CSA)Ea transgene, each with a unique transgene insertion site, were generated and studied (1196D, 1197C, 1199E, and 1200D). Similarly, four unique transgenic lines carrying hGH/BAC were established and studied (1210B, 1252B, 1253E, 1254D). Total RNAs were purified from the transgenic placentas of the indicated lines and subjected to Northern blot analysis. The transgene copy number in each line is indicated at the bottom of each lane. The probes for 18S rRNA and mouse placental lactogen II (PLII) were used as controls. Although robust and copy-number-dependent hGH-hCS expression was observed for all four hGH/BAC lines, no signals were detected for any of the HSIII-V/Pa(CSA)Ea lines at the level of Northern blot sensitivity. (C) Comparison of the hCS-A expression levels in transgenic placentas. RT-PCR analysis was performed to amplify the hCS-A and hCS-B mRNAs using total RNAs isolated from placentas of the indicated transgenic lines. The signal intensity for hCS-A was normalized to that of β-actin as well as to the transgene copy numbers noted in panel B. The calculated relative expression levels are plotted in the frame below. The hGH/BAC lines showed strong site-of-integration-independent and copy-number-dependent expression of hCS-A in placenta. In contrast, expression in the HSIII-V/Pa(CSA)Ea lines was quite low and showed marked position effects. (D) The HSIII-V/Pa(CSA) gene failed to retain placental specificity. Northern blot analysis was performed with RNAs prepared from the indicated tissues. The tissues were isolated from the lines with the highest transgene copy number for each construct. Line 1251F (hGH/BAC) had 35 copies of the transgene; line 1196C (HSIII-V/Pa(CSA)Ea) had 63 copies. The oligoprobe for 18S rRNA was used as a loading control. hCS-A expression is predominantly placenta specific in the hGH/BAC line but is ectopically expressed at high levels in kidneys of the HSIII-V/Pa(CSA)Ea mice.