Genetically manipulated mouse models of lung disease: potential and pitfalls

Abstract

Gene targeting in mice (transgenic and knockout) has provided investigators with an unparalleled armamentarium in recent decades to dissect the cellular and molecular basis of critical pathophysiological states. Fruitful information has been derived from studies using these genetically engineered mice with significant impact on our understanding, not only of specific biological processes spanning cell proliferation to cell death, but also of critical molecular events involved in the pathogenesis of human disease. This review will focus on the use of gene-targeted mice to study various models of lung disease including airways diseases such as asthma and chronic obstructive pulmonary disease, and parenchymal lung diseases including idiopathic pulmonary fibrosis, pulmonary hypertension, pneumonia, and acute lung injury. We will attempt to review the current technological approaches of generating gene-targeted mice and the enormous dataset derived from these studies, providing a template for lung investigators.

Fig. 1.

Generation of knockout mice. A vector is constructed containing homologous regions of the gene targeted for deletion (Homologous, in gray) that surround a drug selection marker such as the neomycin resistance cassette (Neo-R). This vector is then introduced into embryonic stem cells in culture (e.g., by electroporation), where homologous recombination with the endogenous gene of interest occurs, resulting in a subset of cells containing the recombined product containing the disrupted endogenous gene (gene fragments, or Gene Frag) and the drug selection marker. Drug selection is then performed to isolate the desired clone, which is then microinjected into a blastocyst that is then transferred to the uterus of a surrogate mother, with the goal of obtaining germ line transmission and offspring harboring the genetic modification. (Adapted from Ref. with permission).

Fig. 2.

Approaches in generating knockout and transgenic mice. Shown here are some examples of strategies in generating knockout and transgenic mice with tissue-specific and conditional expression of genetic modifications. In 1 type of conditional expression system, a mouse containing a tissue-specific promoter (TSP) driving Cre-recombinase can be bred to mice harboring targeted regions for deletion flanked by loxP sites, with resultant offspring harboring tissue-specific expression of the recombined genetic material. This strategy can be used to conditionally delete a gene target in knockout mice (KO) or overexpress a gene target in a transgenic mouse (TG) by deleting a genetically engineered Stop codon, thus allowing the promoter (Pro) to transcribe the gene of interest (see KO-Conditional and TG-Conditional examples in figure). Another type of conditional expression system involves engineering a TSP to drive rtTA (reverse tetracycline-controlled transactivator) that, when combined with a tetracycline-response element (TRE) in the presence of doxycycline, permits TSP and doxycycline (Dox)-dependent gene expression (see TG-Conditional example in figure). Dox-dependent Cre-recombination can also be undertaken, as in the KO-Conditional Tet-on example in the figure, thus employing multiple levels of complexity in controlling gene expression. [Adapted with kind permission from Springer Science+Business Media: Aoki K, Taketo MM. Tissue-specific transgenic, conditional knockout and knock-in mice of genes in the canonical Wnt signaling pathway. In: Wnt Signaling Volume 1: Pathway Methods and Mammalian Models, edited by Vincan E. Totowa, NJ: Humana, 2008, vol. 468, chapt. 24.].