SELECTED EXAMPLES: MURINEMODELS
OF HUMAN DISEASE
Genetic and Genomic Approaches to
Complex Lung Diseases Using Mouse Models
Michael J. Holtzman, Edy Y. Kim, and Jeffrey D. Morton
1. INTRODUCTION
Common lung diseases are likely to be multifactorial and multigenic. In
addition, the lung exhibits a limited set of biological and physiological
responses, so different lung diseases exhibit significant overlap in
phenotype. This complexity in the development and manifestation of
lung disease poses significant challenges for developing complete and
accurate models of disease. Nonetheless, a layered strategy that includes
in vitro and in vivo systems can offset these limitations. In vitro systems
have evolved from simple organ culture to intricate procedures for cell
culture that exhibit high fidelity to behavior in vivo. Similarly, in vivo
systems have evolved from traditional physiology-based models in large
animals and rodents to genetic modification of mice using targeted and
conditional systems. Complex traits may be studied in inbred,
recombinant, or congenic strains of mice, and single gene effects may be
segregated naturally or experimentally. Ultimately, results from these
in vitro and in vivo models identify candidate genes for further study in
humans.This chapter reviews the development and application of
genetic and genomic approaches to complex lung diseases, focusing on
the use of mouse model systems. Although the particular strength of the
murine system has been its applicability to studies conducted in vivo,
suitable cell culture systems have now been established for comparative
work in vitro as well.Whenever possible, the extension and correlation
of findings to human studies will also be noted. The chapter is organized
by disease entity, using the examples of cystic fibrosis (CF), emphysema,
tuberculosis, and asthma as illustrative of well-studied targets for
multidisciplinary genetic and genomic 103 From: Computational
Genetics and GenomicsEdited by: G. Peltz © Humana Press Inc.,
Totowa, NJ approaches. In addition, the distinct characteristics of these
diseases have shaped different approaches to defining underlying
genetic mechanisms and so illustrate the range of available methods
that can be used in mouse models of lung disease.
2. CYSTIC FIBROSIS
CF is the most common autosomal-recessive disorder in Caucasian
populations, with an incidence of 1 in 2500 live births (1). Thus, the
identification of the CF transmembrane conductance regulator (CFTR)
as the site of the underlying genetic abnormality for this disease was a
seminal example of the positional cloning approach to identify and
characterize a candidate gene in humans (2–4). The CFTR gene has 27
exons that span 230 kb of the long arm of human chromosome 7
(7q31.2) and encodes a transmembrane glycoprotein of 1480 amino acid
residues (5). The gene product is a member of the adenosine
triphosphate-binding cassette family of transporters that have
conserved transmembrane and nucleotide-binding domains linked by a
regulatory domain with phosphorylation sites for protein kinases Aand
C (2,6). Before the identification of the CF TR gene, physiological
studies showed that CF epithelia have defective cyclic adenosine
monophosphate MP)-mediated chloride ion transport (7–9). Subsequent
studies indicated that CFTR was the major cAMP-regulated chloride
channel in the apical membrane of epithelial cells (10,11) and so had a
central role in transepithelial salt transport, fluid flow, and ion
concentrations in the intestine, pancreas, sweat gland, and airway
epithelia (12). CF results from defective CFTR activity that disrupts
transepithelial ion transport. In general, nonsense or stop mutations in
CFTR result in severe disease, whereas missense mutations result in
milder disease. More than 900 mutations have been identified within
human CFTR. The most frequent CF mutation includes 66% of mutant
CFTR alleles. This mutation deletes in-frame the phenylalanine at
position 508 (F508) (13). The F508 CFTR allele produces a
misfolded protein that is trapped in the endoplasmic reticulum (14–16).
In contrast,another common mutation (G551D) results in a protein with
normal processing but decreased chloride channel activity (17).
2.1. Murine Models of CF Anumber of mouse models of CF were
developed based on targeted mutations of the Cftr gene (Table 1). Initial
strategies were based on knockout technology that introduces a genomic
construct into mouse embryonic stem cells. Clones that have undergone
genetic recombination are implanted into pseudopregnant female mice.
The resulting chimeric mice are crossed to 104 Holtzman et al. Genetic
and Genomic Approaches to Complex Lung Diseases 105 produce mice
that are homozygous for the targeted gene deficiency. The first mouse
model disrupted the CFTR gene by introducing a stop codon in exon 10
(strain CFTRm1UNC) (12,18,19). These CFTR-deficient mice are viable
but lack the lung inflammation found in human CF. Instead, these mice
develop severe bowel disease that was fatal by 40 d of age. A second
gene knockout model (CFTRm1HGV) used insertional mutagenesis
rather than the strategy of gene replacement used for the
CFTRm1UNC strain. The CFTRm1HGV mouse strain produces 10%
of normal CFTR levels and has 90% long-term survival with
abnormalities of the colon and vas deferens (20). The CFTRm1UNC
strain has been complemented by human CFTR driven by the rat
intestinal fatty acid-binding protein promoter (21). However, like the
CFTRm1UNC strain