Summary
Previously characterized rat WAP 5' and 3' elements were used to target expression of a
human SP-B genomic sequence to the mammary gland in mice. Expression of rWAP/SP-B mRNA was
specific to mammary gland and ranged from 1 to 5% of the endogenous WAP mRNA levels. SP-B
was detected immunolgically in both tissue and milk. The transgene product had an apparent
molecular weight of 40-45 kDa, corresponding to the predicted size of the SP-B proprotein.
Transgenic females lactated normally and weaned pups without incident.
Background
Rat WAP elements previously characterized as causing tissue specific, hormonally
regulated, copy number dependent transgene expression were employed to direct expression
of SP-B to the mammary gland. SP-B was chosen as a transgene due to its potential
importance in treating both respiratory distress syndrome (RDS) and adult respiratory
distress syndrme (ARDS) patients. RDS affrects approximately 65,000 premature and low
birthweight infants in the U.S.A. and Canada each year. ARDS affects approximately 500,000
adult patients each year in the same area. Both syndromes are characterized by high
morbidity and mortality. Recent studies indicate that surfactant replacement mixtures
containing protein perform better clinically. Experiments in a premature rabbit model
imply that SP-B is the primary factor in this improved performance. We therefore undertook
to produce SP-B in milk in order to provide a source of the material at low cost. We
envision it as a component in a reconstituted surfactant mixture with enhanced clinical
efficacy.
Key Citation
S, Yarus, N.M. Greenberg, Y. Wei, J. A. Whitsett, T. E. Weaver, and J. M. Rosen (1996)
Secretion of Unprocessed Human Surfactant Protein B in Milk of Transgenic Mice Transgenic
Research 5: (in press)
Transgene
The rat WAP promoter (-949 to + 32 with respect to transcription, see Genbank: X01153) was
cloned into the XbaI-SpeI site of a Bluescript II SK+ (Stratagene) vector as a 980 bp Hinc
II-SpeI fragment . Human genomic SP-B sequences corresponding to bp 1035-7770 of Genbank:
M24461 (exons 1-10 of hSP-B) were cloned 3' to the promoter. An 843 bp ClaI-XhoI PCR
fragment encompassing a portion of the third exon of rWAP to 70 bp past its
polyadenylation site was subsequently ligated into the Pvu II site, at 7770 in the SP-B
sequence M24461. This generated the final construct designated WAP/SP-B.
Mouse Strain : ICR
Phenotype
The phenotype is best characterized as benign. No effects on morphology or lactation were
observed. SP-B proprotein was detected throughout the gland, both in milk secreted into
the lumen and on the apical border of cells surrounding the lumen. Stromal staining was
weak and did not correlate to presence of the transgene or the primary antibody employed.
Phenotype Image
Transgenic WAP/SP-B mice show no changes in gross morphology
Histology
Figure 1: Mammary gland at 10 days lactation from a WAP/SP-B transgenic mouse. Anti SP-B sera is primary antibody (100X).
Figure 2: As in Figure 1, with tissue from nontransgenic sister.
Figure 3: As in Figure 1 but primary antibody is commercial normal rabbit sera (Sigma Chemical).
All figures are 5 micron paraffin sections probe with primary antibody as indicated. Detection of antibody binding was accomplished with the Vectastain ABC kit (Vector Labs, Burlington CA). Methylene green was employed as a counterstain.
Mammary Development
Transgenic females lactated normally and weaned normal sized litters without incident.
Some transgenic females were bred repeatedly without ill effect.
Gene Expression
Transgene expression was detected only in lactating mammary tissue despite an extensive
organ survey. Levels were 1-5% of endogenous mouse WAP. Two lines were characterized. A
third line did not express, apparently because it contained no intact copies of the
transgene.
Mechanistic Implications
Apparently the mammary gland lacks proper enzymes for post translational cleavage of SP-B
which is normally produced in type II alveolar pneumocytes. Cathepsin D, which had been
implicated in processing SP-B at the time this project was undertaken did not cleave the
aminoterminal domain of SP-B in the mammary gland. This result dictates an alternative
strategy for producing processed SP-B by using a modified genomic construct.
Submitted by
Sinai Yarus on February 11, 1996
Dept. of Cell Biology, Baylor College of Medicine, Houston TX, 77030, USA
Phone : (713)-798-6211
Fax : (713)-798-8012
Contact : syarus@mbcr,bcm.tmc.edu
Keyword
contributed March 1996 |
