Sinai Yarus¹, Timothy E. Weaver², and Jeffrey M. Rosen¹

¹ Department of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030-3498, USA

² Division of Neonatology and Pulmonary Biology, University of Cincinnati College of Medicine and Children's Hospital Medical Center, Cincinnati, Ohio 45267-0541, USA

Received 3/24/97; Accepted 4/22/97; On-line 5/1/97

4. RESULTS

Expression of SP-BDELTAC mRNA in transgenic mice

Five transgenic founders carrying the WAP/hSP-B DELTA C transgene were identified by PCR analysis. Founders 4927, 4928 and 4931 were female while 4934 and 4945 were male. Biopsies of the female founders were performed and only line 4928 expressed SP-B DELTA C RNA at a level detectable by Northern blot analysis (Fig. 1A). However, the other two female lines 4927 and 4931 did not transmit the transgene to any of their progeny suggesting that they were mosaic founders. These two lines were not characterized further. Female F1 progeny of the male founder lines 4934 and 4945 both expressed the SP-B DELTA C transgene, although at variable levels (Fig. 1B). RNA samples from line 4934 required a 5 day exposure to X-ray film for detection of the SP-B DELTA C transcript, while SP-B mRNA from lines 4928 and 4945 samples was visualized after only a one day autoradiographic exposure. Transcript levels in the 4945 line were approximately 2.5 times greater than those in the 4928 founder but this difference was not statistically significant due to variation within the 4945 line. Since expression of "housekeeping" genes is affected by lactation, and expression of transgenes in lactating mammary tissue may disrupt expression of endogenous milk genes, equality of loading was judged from Ethidium Bromide staining of gels (Fig.1 A and B) using multiple inputs of total RNA. All transgenic mice expressing the SP-B DELTA C transgene displayed two transcripts (Fig. 1). The predominant mRNA corresponds to the expected transcript size of 1019 bp, while the minor transcript had an apparent size of 1800 bp perhaps due to inappropriate post-transcriptional processing.

Figure 1: Northern blot analysis of WAP/SP-B lines. 10, 5, and 1 µg of total RNA from a mammary gland on day 10 of lactation were subjected to Northern blot analysis using a human SP-B probe (810 bp). A) Female founders 4927, 4928 and 4931. 4928 express the predicted 1019 bp transcript as well as an 1800 bp transcript. (overnight exposure) B) F1 females from the [4934] and [4945] lines all express the predicted 1019 bp transcript as well as a 1800 bp transcript (exposure time is 5 days for [4934] and overnight for [4945]).

SP-BDELTAC protein in milk

Western blot analysis of milk from female 4928 showed the predicted 28 kDa protein (Fig. 2A) as well as a slightly smaller species. The 28 kDa SP-B D C was detected with antibodies R28031, 55019 and R55522, but not with R96189. This confirmed that the 28 kDa protein contains the amino-terminal and active peptide domains, but not the carboxy-terminal domain. A sample of milk from a SP-B transgenic female containing the previously characterized proprotein (42 kDa) (1) reacted with all four antibodies. SP-B proprotein in mouse lung tissues is rapidly processed to the mature peptide (8 kDA) and consequently the precursor protein is not detected (Fig. 2B, lane L).

Figure 2: Western blot analysis of mouse milk and lung extracts. A) 1.0 µl milk/lane was subjected to PAGE and blotted to PVDF membrane (1; 4928), (2; 4931), (3; non-transgenic sample), (4; milk from a transgenic full length SP-B mouse). The blots were probed with the polyclonal rabbit sera (1:700 in TBS + 5% NFDM) with specificities to different domains of the SP-B proprotein. Sera and their specificity are indicated below each portion of the blot. Visualization of the antigen antibody complex was accomplished with biotinylated goat anti-rabbit serum, streptavidin-horseradish-peroxidase, and ECL reagents. B) Samples treated as in A. L= 25 µg of total lung homogenate and M= 1.0µl of milk from an F1 female of the [4945] line.

Comparison of milk from SP-BDELTAC transgenic mice to SP-B derived from lung tissue (Fig. 2B) confirmed that the fully processed SP-B did not appear in the milk as a result of the carboxy-terminal truncation. The predicted 28 kDa species is clearly the major product of the transgene, although other bands are apparent, either as a result of proteolysis, or cross-reactivity of the primary antibodies with unrelated proteins in the milk.

Phenotype of line 4945

While females from lines 4928 and 4934 nursed normal-sized litters to weaning without incident, females of line 4945 routinely cannibalized all or part of their litters. Surviving pups from this line were retarded in growth, although they grew to normal size after weaning. Histochemical analysis of mammary gland tissue isolated on day 10 of the second lactation from 4945 F1 females (Fig. 3) revealed both abnormally low amounts of secretory epithelium and an altered morphology of individual alveoli. Mammary gland isolated from day 1 of lactation from 4945 F1 (third lactation) and F2 (first lactation) females showed a similar pattern (Fig. 4).

Figure 3: Histochemical analysis of mammary tissue in mid-lactation shows retarded development. Paraffin-embedded sections of normal (A and C) and transgenic line 4945 (B and D) mammary gland on day 10 of lactation were stained with haemotoxylin and eosin. (A and C) 40x (B and D) 400x .

Figure 4: Histochemical analysis of mammary tissue in early lactation shows retarded development. Paraffin-embedded sections of transgenic mammary gland on day 1 of lactation were stained with haemotoxylin and eosin. All tissues are from [4945] line: (A, B);tissue from an F1 female in third lactation, (C, D); tissue from an F2 female in first lactation. (A and C) 40x (B and D) 400x

The affected alveoli lack a clearly defined lumen and appeared to be surrounded by multiple epithelial cell layers. In addition, the amount of connective stromal tissue, relative to secretory epithelial tissue was greatly increased.