FIGURE 6.

Southern blot analysis of transgene integration in hTG mice. Genomic DNAs were prepared from hTG mice (SP-A1(6A⁴) and SP-A2(1A³)), SP-A KO mice, and WT mice as well as from human lung tissue. The DNA samples were digested with EcoRI. The digested DNAs were subjected to agarose-gel electrophoresis and then transferred to a nylon membrane. The hSP-A1 or hSP-A2 DNA bands were detected with ³²P-labeled DNA probes of 0.6-kb or 1.1-kb fragments of human SP-A cDNA. The linearized plasmid DNA (about 8 kb, pointed out by an arrow) carrying the hSP-A cDNA (described in the legend to Fig. 1) was used as a control. After hybridization with either probe, the blot membrane was washed three times (see “Materials and Methods”) and then exposed to XAR film. A and B depict the RFLP pattern of hSP-A as detected by the 0.6- and 1.1-kb probes from SP-A cDNA, respectively. The 0.6-kb probe is located within the coding region (exons I–IV) of SP-A cDNA. This region contains no EcoRI cleavage site in either SP-A cDNA or human genomic DNA. The 1.1-kb probe contains the coding region (exons I–IV) and a partial 3′-UTR sequence (present in exon IV) of SP-A cDNA with one EcoRI cleavage site. This site is in the 3′-UTR of SP-A cDNA and human genomic DNA. The data from A indicate that there are two DNA bands (5.8 and 5.1 kb, marked by ▶ and >, respectively) in each of the hTG mice, SP-A1(6A⁴) and SP-A2(1A³). The human genomic DNA (hDNA) also shows two DNA bands (7.7 and 6.3 kb, marked by ● and ●●, respectively); the 6.3-kb fragment is from SP-A1, and the 7.7-kb fragment is from SP-A2. The RFLP pattern of B with the 1.1-kb probe shows that the hTG mice (SP-A1(6A⁴) and SP-A2(1A³)) also contained two DNA bands (5.8 and 5.1 kb, marked by ▶ and >, respectively), as shown in A. Human genomic DNA contains four bands. Of these, the 6.3 kb and 4.3 kb bands (marked by ●● and ○, respectively) are from SP-A1, and the 7.7 kb and 2.2 kb bands (marked by ● and ○○, respectively) are from SP-A2.