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

3. METHODS AND MATERIALS

Oligonucleotides

The oligonucleotides used in this study are shown in Table 1.

Table 1: Oligonucleotides Summary of names and sequences of oligonucleotides employed in PCR, sequencing and cloning are presented in table 1.

5'-CAT GGA TTA ACT GAC TAG CGC GC-3'

5'-CAT GGC GCG CTA GTC AGT TAA TC-3'

5'-CTA CTC CGT CAT CCT GCT CGA C-3'

5'-CTC ACT GAA AGG ATA TCA CTG TAG GAG A-3'

5'-ATC AGT CAT CAC TTG CCT GCC GCC G-3'

5'-TGA CCA GGG GGA AGT AGT CGT-3'

Construction of pWAP/SP-BDELTAC

pHga33 was prepared by excising an HgaI fragment containing the SP-B sequence from pWAP/SP-B corresponding to gb:m24461 (4230-7700) (1) and subcloning it into the EcoRV site of pBluescript II (Stratagene, La Jolla, CA). pHga33 contained a single NcoI site [gb:m24461 (5546) ], which is situated near the junction between the SP-B processed peptide and carboxy-terminal domains. Two complementary oligonucleotides (NcoStop Forward and NcoStopReverse; Table 1) were designed so that they could be annealed (10) to form NcoI compatible protruding 5' termini. In addition, the annealed oligonucleotides restored the final Asp residue of the SP-B processed peptide domain, contained stop codons in each of the 3 reading frames, and introduced a complete BssHII restriction endonuclease site 3' to the newly introduced stop codons.

pHga33 was digested with NcoI and the annealed NcoStop oligos were added by ligation to produce pHga33Stop. pHga33Stop and pWAP/SP-B were both then digested with BsmBI. The 2325 bp BsmBI fragment from pHga33Stop, containing the newly introduced stop codons, was ligated directly into the pWAP/SP-B vector. Since BsmBI cuts adjacent to its recognition site, this was a directional cloning step. The resultant pWAP/SP-B(Stop) clones were screened initially by BssHII digestion, and their identity was confirmed by sequencing using the SPBex7Forward oligonucleotide (Table 1) as a primer.

After confirming the presence of the NcoStop oligos and the preservation of the reading frame at the ligation junction by sequencing, the WAP promoter and SP-B(Stop) sequence encoding the amino-terminal and processed peptide domains were excised as a BssHII fragment, treated with Klenow (10), and then digested with Not I. The WAP 3' vector pWE3' (9) was digested with Not I and EcoRV, permitting directional cloning of the WAP promoter and SP-B(Stop) fragment to create the final construct pWAP/SP-BDELTAC.

Generation and screening of transgenic mice

The transgene was excised as a 6.5 kb Bssh II fragment, purified by adsorption to Qiaex II resin (Qiagen, Chatsworth, CA) and subjected to micro-injection (11). Genomic tail DNA was analyzed by PCR screening as previously described (1). PCR was conducted for 30 cycles of 1 min at 94°C, 2 min at 60°C, 3 min at 72°C and samples were held at 4°C until analysis. The forward primer was WAP +1Forward and the reverse primer was SP-Bex2Reverse (Table 1)

RNA preparation and Northern blot analysis

Mammary gland biopsies were performed on the female founder mice and the F1 females in mid-lactation under anesthesia (9). Total RNA was isolated by RNAzol (TEL-TEST , Friendswood, TX) according to the manufacturer's instructions. Fractionation of total RNA was on agarose gels containing formaldehyde (10). After transfer to ZetaProbe GT membrane (Bio-Rad, Hercules, CA), RNA was hybridized to ³²P-labeled human SP-B fragments generated using random hexanucleotide primers (10). Transfer, hybridization and filter stripping were performed according to the manufacturer's recommendations. Quantitation was performed using a Phosphorimager (Molecular Dynamics, San Francisco, CA) and the relative expression levels of SP-BDELTAC mRNA of transgenic lines were estimated.

Antibodies

Four polyclonal rabbit sera (previously prepared by TW) were employed in immune detection of western blots (2). R28031 reacts exclusively with epitopes in the fully processed SP-B molecule. R55522 reacts to epitopes in both the amino-terminal and the carboxy-terminal domains of the SP-B proprotein. R55019 reacts to epitopes only in the amino-terminal domain of the SP-B proprotein. R96189 reacts to epitopes only in the carboxy-terminal domain of the SP-B proprotein. These antibodies were employed in combination to determine which domains of the SP-B were produced by the transgene.

Lung homogenates

Lung homogenates were prepared for Western blot analysis as previously described (12) except that the primary antibodies were those described above.

Milk collection, PAGE, and Western blotting

Milk samples were collected during mid-lactation from anaesthetized mice using a constant vacuum apparatus (13). Polyacrylamide gel electorphoresis (PAGE) was performed according to the method of Schagger and von Jagow (14). 16.5%T/3%C Tris/Tricine gels were run overnight at 70v. Western transfer was for 90 min at 300 mA. Western blot analysis was performed as described previously (9) except that the primary antibodies were those described above. Visualization was by enhanced chemiluminescence (ECL, Amersham, Arlington Hts., IL)

Histochemistry

Mammary gland biopsies were obtained from anaesthetized mice on day ten of lactation. Mice were separated from their pups for 3 hr prior to biopsy to insure a buildup of milk in the alveoli. Tissue samples were fixed in 10% neutral buffered formalin for 8 hr at room temperature. Samples were rinsed twice in large volumes of deionized water and stored in 70% ethanol until embedding. Cubes of tissue from the central portion of the gland were dehydrated and embedded in paraffin. Sections (5 µm) were mounted on Probe-On Plus (Fisher Scientific, Pittsburgh, PA) slides. Sections were deparaffinized and rehydrated through an ethanol series prior to staining with hemotoxylin/eosin.