Transgenic mouse models of human lung diseases

Abstract

Transgenic mice can provide useful probes into complex biological systems. We have produced transgenic mice expressing human neutrophil elastase (HNE) in their lungs in order to better understand the elastase-antielastase theory and produce an animal model for emphysema, as well as mice expressing the human Krev-l in order to test its activity in vivo.

Disruption of the protease anti-protease balance in the lung is believed to play a key role in the aetiology of emphysema. In an attempt to produce an animal model for emphysema, we have generated transgenic mice by microinjection of HNE genomic DNA under the control of the rabbit uteroglobin (UG) promoter. Eight independent transgenic lines were generated. Analysis of total RNA by Northern hybridisation showed strong expression of HNE mRNA in transgenic lung of 6 out of 8 lines, and weak expression in a seventh line. Immuno histochemistry using specific anti-HNE antibodies indicated the presence of HNE protein in the bronchial epithelium. The protein was detected immunologically in bronchoalveolar lavages, indicating that HNE protein was secreted. On Western blotting of lavage fluid, a biotinylated anti-HNE antibody detected two bands of approximately 30,000 and 80,000 Mr corresponding to free and complexed HNE, respectively. No HNE activity was detected in the bronchoalveolar lavages of transgenic mice. Examination of mouse lung sections occasionally revealed mild signs of emphysema but there was no difference in incidence of emphysema between transgenic and wild-type groups.

Mice of the A/J strain are useful models of lung cancer because they develop tumours spontaneously or after treatment with ethyl carbamate. These tumours are thought to arise either from the Clara cells (papillary tumours) or from the alveolar type 2 cells (alveolar tumours), and, like many human lung adenocarcinomas, involve K-ras activation. Transformation with K-ras can be reversed by co-expression of the Krev-l gene in tissue culture. To test the tumour suppressor activity of Krev-l in vivo we have produced transgenic A/J mice expressing Krev-l in their lungs under the control of the rabbit uteroglobin promoter. Sixty-six mice (35 transgenic and 31 non-transgenic) from 3 lines were given ethyl carbamate and the numbers of lung tumours were compared between transgenic and non-transgenic animals. The mean number of ethyl carbamate-induced lung tumours was 21.7 ± 1.3 (s.d.) in transgenic mice and 26.9 ± 1.6 in non-transgenic littermates (p<0.01). These data demonstrate the activity of Krev-1 in vivo. In a second experiment, 8 mice (3 transgenic and 5 non-transgenic) were given ethyl carbamate and the resulting tumours were dissected and examined microscopically. The numbers of alveolar tumours were 4.7 per transgenic mouse and 3.8 per non-transgenic mouse, whereas the numbers of papillary tumours were 5.3 per transgenic mouse and 6.8 per nontransgenic mouse. As UG-driven trans genes are expressed in Clara cells, this reduction in the percentage of papillary tumours in transgenic mice is consistent with the Clara cell origin of at least some of the papillary tumours.

During the course of this work, a plasmid with a protruding G at each end that can be used as a positive control for end-filling prior to blunt-end ligation was developed.

We also developed a simple method for efficient blunt-end ligation. We have shown that construction of recombinant plasmids can be done efficiently if ligation is first carried out at high DNA concentration, to favour intermolecular events, then the DNA is diluted to low concentration to favour intramolecular events. Using this method, the number of recombinant plasmids obtained was about 10 times higher than in the control ligation with the generally recommended DNA concentration.