Comparative biology of cystic fibrosis animal models

Abstract

Animal models of human diseases are critical for dissecting mechanisms of pathophysiology and developing therapies. In the context of cystic fibrosis (CF), mouse models have been the dominant species by which to study CF disease processes in vivo for the past two decades. Although much has been learned through these CF mouse models, limitations in the ability of this species to recapitulate spontaneous lung disease and several other organ abnormalities seen in CF humans have created a need for additional species on which to study CF. To this end, pig and ferret CF models have been generated by somatic cell nuclear transfer and are currently being characterized. These new larger animal models have phenotypes that appear to closely resemble human CF disease seen in newborns, and efforts to characterize their adult phenotypes are ongoing. This chapter will review current knowledge about comparative lung cell biology and cystic fibrosis transmembrane conductance regulator (CFTR) biology among mice, pigs, and ferrets that has implications for CF disease modeling in these species. We will focus on methods used to compare the biology and function of CFTR between these species and their relevance to phenotypes seen in the animal models. These cross-species comparisons and the development of both the pig and the ferret CF models may help elucidate pathophysiologic mechanisms of CF lung disease and lead to new therapeutic approaches.

Fig. 19.1

Tracheal xenograft design and transplantation. (a) The cassette is composed of flexible plastic tubing, freshly excised newborn pig or ferret trachea, and chrome wire plugs (see Note 1). The trachea is fastened to the tubing using silk sutures. (b) The xenograft cassettes are inserted subcutaneously into the flanks of Nu/Nu athymic mice. The xenografts vascularize within 2–3 weeks and continue to mature and develop until ready for bioelectric characterization by measuring PD (4–5 weeks). PD recordings are made weekly until 8–9 weeks post-transplantation. Usually a CFTR^−/− xenograft is transplanted in parallel to either a CFTR^+/− or a CFTR^+/+ xenograft.

Fig. 19. 2

Potential difference instrumentation and setup. Measuring TEPD in this ex vivo model requires the following equipment: computer with data acquisition software, pH/mV meter, calomel electrodes, and syringe pump. The pH/mV is connected to the calomel electrodes that are connected to the anesthetized mouse by means of butterfly electrodes (see Note 2). The positive electrode is inserted into the perfusion tubing (black arrows) allowing access to the luminal surface of the trachea, while the negative electrode is inserted subcutaneously (white arrows).

Fig. 19.3

TEPD analysis of ferret CF and non-CF tracheal xenografts. (a) Representative TEPD tracings of newborn ferret CFTR^+/+ (dark line) and CFTR^−/− (light line) tracheal xenografts. The buffer conditions change with ion channel agonists and antagonists are indicated above the tracing. (b) Reproducibility of sequential TEPD measurements taken in the same ferret CFTR^+/+ and CFTR^−/− xenografts at week intervals as indicated. Buffer conditions were the same as shown in (a) with the buffer number marked arrowheads. (c) Histological H&E sections of ferret CFTR^+/+ (top) and CFTR^−/− (bottom) xenografts. Note the intact pseudostratified ciliated epithelium (empty arrows) and the presence of submucosal glands (solid arrows) in both genotypes.

Fig. 19.4

Metabolic [³⁵S]methionine pulse chase of ferret CFTR processing. (a) HT1080 cells transiently expressing ferret CFTR were starved of methionine and cysteine (30 min), labeled with [³⁵S]methionine and [³⁵S]cysteine (15 min), and chased with media containing cold methionine and cysteine for the given time points as described under Section 3.3. (b) Densitometric quantification of (a) Empty points representing band B_T/band B₀ × 100. Solid data points representing band C_T/band B₀ × 100 (see Note 13).