pulmonary-specific gene expression in transgenic mice - Europe PMC

Proc. Nati. Acad. Sci. USA Vol. 87, pp. 6122-6126, August 1990 Developmental Biology

Cis-acting sequences from a human surfactant protein gene confer pulmonary-specific gene expression in transgenic mice (cell ablation/lung cell development/lung-specific expression)

THOMAS R. KORFHAGEN, STEPHAN W. GLASSER, SUSAN E. WERT, MICHAEL D. BRUNO, CYNTHIA C. DAUGHERTY, JOHN D. MCNEISH, JEFFREY L. STOCK, S. STEVEN POTTER, AND JEFFREY A. WHITSETT* Children's Hospital Medical Center, Department of Pediatrics, Elland and Bethesda Avenues, Cincinnati, OH 45229

Communicated by Richard Palmiter, May 29, 1990

9). However, cis- or trans-active elements controlling lungspecific and developmental expression of the surfactant proteins have not been identified. Human SP-C is a 3700-Da hydrophobic peptide derived from a 21,000-Da precursor (3, 8). Synthesis of SP-C mRNA has been detected only in the lung (3, 10), appears in late gestation in the rat (11), and is detected at 13 weeks in the human fetal lung (12). The entire nucleotide sequence of the human SP-C gene has been recently reported from this laboratory; the gene is -2.6 kilobases (kb) in length and consists of six exons and five introns located on chromosome 8 (8). Fusion genes consisting of the diphtheria toxin A (DT-A) gene controlled by tissue-specific enhancer/promoter sequences have been used to ablate ocular lens (13, 14), pancreatic cells (15), and pituitary cells (16) in transgenic mice (for review, see ref. 17). To determine whether cisactive elements in the human SP-C gene might determine lung-specific gene expression, we have constructed a chimeric gene consisting of the human SP-C promoter and its 5'-flanking sequences with a bacterial DT-A gene and generated transgenic mice containing the fusion gene.

Pulmonary surfactant is produced in late ABSTRACT gestation by developing type I epithelial cells ling the alveolar epithelium of the lung. Lack of surfactant at birth is associated with respiratory distress syndrome in premature infants. Surfactant protein C (SP-C) is a highly hydrophobic peptide isolated from pulmonary tissue that enhances the biophysical activity of surfactant phospholipids. Like surfactant phospholipid, SP-C is produced by epithelial cells in the distal respiratory epithelium, and its expression increases during the latter part of gestation. A chimeric gene containing 3.6 kilobases of the promoter and 5'-flanking sequences of the human SP-C gene was used to express diphtheria toxin A. The SP-C-diphtheria toxin A fusion gene was injected into fertilized mouse eggs to produce transgenic mice. Affected mice developed respiratory failure in the immediate postnatal period. Morphologic analysis of lungs from affected pups showed variable but severe cellular injury confimed to pulmonary tissues. Ultrastructural changes consistent with cell death and injury were prominent in the distal respiratory epithelium. Proximal components of the tracheobronchial tree were not severely affected. Transgenic animals were of normal size at birth, and structural abnormalities were not detected in nonpulmonary tissues. Lung-specific diphtheria toxin A expression controlled by the human SP-C gene injured type U epithelial cells and caused extensive necrosis of the distal respiratory epithelium. The absence of type I epithelial cells in the most severely affected transgenic animals supports the concept that developing type U cells serve as precursors to type I epithelial cells.

MATERIALS AND METHODS Bacterial Strains and Restriction Endonucleases. Plasmid maintenance and construction were done using Escherichia coli JM101 as host strain. pUC plasmids and restriction endonucleases were purchased from New England Biolabs. Construction of SP-C-DT-A-hGH. Fig. 1 diagrammatically represents the human SP-C gene and plasmid used. DT-AhGH is a plasmid constructed by cloning the DT-A cassette into E.2-hGH, where hGH represents human growth hormone (15), after removal of the elastase promoter. A HindfIlEcoRI fragment was then subcloned into pUC18 to make pUC18-DT-A-hGH. The 5' HindIII-Kpn I fragment of A VG519 was generated by digestion of pSp-CH/H SVOCAT. This latter plasmid contains a 4.4-kb HindIfl-HindIll fragment of AVG519. The 3' Kpn 1-HindIll fragment was prepared from pUC18-SP-C-K/P(Kpn I-Pst I), which is a plasmid containing the appropriate Kpn I-Pst I fragment of AVG519. These fragments were ligated at the Kpn I site to generate a 3.8-kb HindIfl-HindIll fragment that contains the SP-C sequences and 8 base pairs (bp) of the pUC Pst I-HindlIl multicloning site. The 3.8-kb HindIll-HindIfl fragment was cloned into the single HindII site of pUC18-DTA-hGH generating SP-C-DT-A-hGH. Generation of Transgenic Mice. Donor eggs were prepared from C57BL/6 x C3H F1 hybrid mice obtained from Charles

Pulmonary surfactant is a complex mixture of phospholipids and proteins synthesized and secreted by specialized type II cells that are confined to the distal respiratory epithelium. In late gestation, the progenitors of type II cells undergo dramatic structural and biochemical maturation accompanied by increased synthesis of pulmonary surfactant, which is required to reduce surface tension at the air-liquid interface. Postnatally, continued differentiation of type II cells is thought to produce type I epithelial cells, which comprise most of the alveolar surface in postnatal animals. Mature type I cells do not synthesize pulmonary surfactant. Genes and cDNAs encoding three distinct surfactant proteins, SP-A, SP-B, and SP-C, have been isolated by workers in this laboratory and others (1-8). The proteins encoded by these genes confer important surface-active properties to phospholipids. Like surfactant phospholipids, these proteins are synthesized by epithelial cells in the distal components of the lung. Expression of surfactant proteins (SP-A, SP-B, and SP-C) increases with advancing gestation (for review, see ref.

Abbreviations: SP-A, -B, and -C, surfactant proteins A, B, and C, respectively; DT-A, diphtheria toxin A. *To whom reprint requests should be addressed at: Pediatrics/ Neonatology Division, University of Cincinnati College of Medicine, 231 Bethesda Avenue, Cincinnati, OH 45267-0541.

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Developmental Biology: Korfhagen et A K

5' H

P

pUC

FIG. 1. Diagrammatic representation of the SP-C-DT-A-hGH fusion gene. SP-C genomic sequences were inserted as described. The partial map of clone AVG519 shows previously described, cloned SP-C sequences (8) and flanking sequences. Exons are represented above the line with translated sequences darkened. The triangle depicts the approximate location of the TATA box. A partial restriction map is shown. H, HindIII; K, Kpn I; P, Pst I; N, Nde I; E, EcoRI. The diagram is not drawn to scale.

River Breeding Laboratories. Methods used for preparation of DNA from tails, Southern blots, and probes have been described (14). Tissue Preparation. For histologic examination, dissected tissue was fixed by immersion in 3.7% (vol/vol) formalin, dehydrated, infiltrated and embedded in paraffin, and stained with hematoxylin/eosin. For transmission EM, dissected tissue was fixed by immersion in 1.0% (wt/vol) tannic acid/2.5% (vol/vol) glutaraldehyde, dehydrated through a series of ethanol and acetone solutions, and infiltrated and embedded with Epon 812. Semi-thin sections were stained with toluidine blue, and pale-gold sections were poststained with 2.5% (wt/vol) aqueous uranyl acetate and Reynold's lead citrate and then viewed and photographed with a JEOL 100 CX electron microscope. RNA Preparation and Analysis. Preparation of total lung RNA and Northern (RNA) blot hybridization were described (8). RNA was transferred to a Nytran filter and prehybridized in 6 x SSPE (1 x SSPE is 0.18 M NaCl/10 mM phosphate, pH 7.7/1 mM EDTA)/10x Denhardt's solution (lx Denhardt's solution is 0.02% polyvinylpyrrolidone/0.02% Ficoll/0.02% bovine serum albumin)/1% SDS/salmon sperm DNA at 100 ,ug/ml at 42°C for 5 hr. Hybridization was as described (8). RESULTS Preparation of the Chimeric Gene and Transgenic Mice. A partial restriction map of human SP-C clone AVG519, which

Proc. Natl. Acad. Sci. USA 87 (1990)

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contains the entire human SP-C gene, is shown in Fig. 1. The sequences contained within the HindIII-Pst I sites were cloned into pUC18 DT-A-hGH to generate the plasmid SP-C-DT-A-hGH as described. The SP-C sequences contained a TATAA box as described (8) and -3.6 kb of 5'-flanking sequence upstream from the untranslated sequences of exon 1. A small portion (8 bp) of the Pst I-HindIII fragment of the multicloning site of pUC was included between the 3' end of the SP-C sequences and the 5' end of pUC18-DT-A-hGH sequences. A 6.9-kb EcoRI-Nde I fragment was prepared for microinjection from the plasmid shown in Fig. 1. Fertilized mouse eggs were injected as described (14). Litters were delivered from surrogate dams by hysterotomy at days 18 and 19 of gestation. Two litters delivered spontaneously on day 20 of gestation. In all, 43 pups were delivered from 12 litters. Pups described below were observed for up to 45 min and sacrificed by cervical transection. Normal, live-born pups weighing >1 g were generally viable. Control pups weighing