PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of wtpaEurope PMCEurope PMC Funders GroupSubmit a Manuscript
 
Am J Respir Cell Mol Biol. Author manuscript; available in PMC Sep 27, 2013.
Published in final edited form as:
PMCID: PMC3785140
EMSID: EMS53283
The a”MAZE”ing world of lung specific transgenic mice
Emma L. Rawlins1 and Anne-Karina Perl2
1The Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, United Kingdom ; e.rawlins/at/gurdon.cam.ac.uk
2Perinatal Institute, Division of Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229-3039; USA ; Anne.Perl/at/cchmc.org
*Correspondence: Anne-Karina Perl PhD Children’s Hospital Medical Center Divisions of Neonatology and Pulmonary Biology 3333 Burnet Avenue Cincinnati, OH 45229-3039 Phone: (513) 636-6084 FAX: (513) 636-7868 ; Anne.Perl/at/cchmc.org
The purpose of this review is to give a comprehensive overview of transgenic mouse lines suitable for studying gene function and cellular lineage relationships in lung development, homeostasis, injury and repair. Many of the mouse strains reviewed in this article have been widely shared within the lung research community and new strains are continuously being developed. There are many useful transgenic lines that work to target subsets of lung cells, but it remains a challenge for investigators to select the correct transgenic modules for their experiment. This review covers both the tetracycline and tamoxifen inducible systems and will primarily focus on conditional lines that target the epithelial cells. We point out the limitations of each strain so investigators can choose the system that will work best for their scientific question. Current mesenchymal and endothelial lines are limited by the fact that they are not lung specific. These lines will be summarized in a brief overview. In addition, useful transgenic reporter mice for studying lineage relationships, promoter activity and signaling pathways will complete our lung specific conditional transgenic mouse-shopping list.
Keywords: transgenic, conditional, lineage tracing, gene expression, lung, mouse
In recent years tremendous progress has been made in understanding the cellular processes underlying lung development, homeostasis and repair. This has been facilitated by the use of lung cell type-specific transgenic mouse lines. Initially lung specific promoters [1-3] were used to constitutively drive transgenes in a cell type specific manner. This mostly resulted in embryonic lethal phenotypes and consequently limited adult studies. The development of conditional transgenic mice allowed the temporal and spatial control of gene expression, overcoming many lethal phenotypes, and allowing the analysis of lung-specific gene knock-outs, identification of progenitor cells, lineage tracing, and studies of progenitor proliferation and differentiation capacity. The most widely used systems are the doxycycline system (tTA and rtTA) and the Cre-LoxP system, but others exist and their use is becoming more widespread. As transgenic mouse strains have been used their strengths, limitations, and the strategies required for optimal experimental design, have become apparent. In this review we will discuss the mouse strains that have been shown to be the most useful for manipulating gene expression in the lung and also highlight areas where new mice would be extremely beneficial for the community.
Cre recombination
Cre-Lox technology was introduced in the 1980s [4, 5] and patented by DuPont Pharmaceuticals. It was successfully applied to mice in 1998 [6]. The technology is based on the ability of the P1 bacteriophage recombinase (Cre) to direct site-specific DNA recombination between pairs of LoxP sites. Such recombination in a “Cre-lox” mouse can permanently either inactivate, or activate, a gene of interest.
Typical Cre-Lox experiments require two transgenic animals: a Cre strain and a LoxP strain. A Cre mouse contains a Cre recombinase transgene under the control of a tissue-specific promoter (Fig. 1A), whereas a LoxP mouse contains two LoxP sites that flank a genomic segment of interest, the “floxed” locus. Depending on the location and orientation of the LoxP sites in a Cre-Lox mouse, Cre recombinase can initiate deletions, inversions, and translocations of the floxed locus [7]. The floxed loci can be designed to allow either permanent inactivation, or activation, of the gene of interest (Fig. 1B). Mutated LoxP sites, which allow recombination between various independent LoxP sites, have been successfully used to rapidly target genes and generate multicolor reporter mice, ‘brainbow’ and ‘confetti’, which are useful reporters for clonal analysis of progenitor cells [8-10].
Figure 1
Figure 1
Schematic of Cre and Tet transgenic systems
The site of Cre activity (cell type specificity) is dependent on the availability of tissue-specific or cell-specific promoters. Moreover, tissue-specific Cre expression can be combined with temporal-specific activity. Time-specific Cre activation can be achieved by combination with the doxycycline system (Fig. 1D), or by the use of Cre fusion proteins. Cell type specific Cre strains have been widely used in the lung for lineage tracing and permanent gene activation or deletion (for example [11, 12]).
A very useful technique is to track the descendents of stem/progenitor cells (lineage-tracing) by crossing a Cre mouse with a reporter mouse strain that permanently expresses a reporter gene following Cre activity. The Z/AP and Z/EG reporter mice were initially used for lineage-tracing experiments [13, 14]. However, studies have shown that these reporters are not expressed in all cell types and can be silenced in adult tissue. The Rosa26 reporter (Rosa26R) variants are now the most widely-used reporter strains available. The ROSAβgeo26 (GtROSA26) line was initially derived from pools of ES cells infected with the retroviral gene trap vector Gen-ROSAβgeo [15]. After cloning the ROSA26 locus [16], it was used to produce a variety of Cre reporter lines starting with lacZ and expanding it to a vast repertoire of cytosolic, membrane bound and nuclear florescent lineage-tags [17]. The ROSA26 locus is particularly useful for generating these strains as it is expressed robustly in most cell types and is gene-targeted at high efficiency, thus numerous other cassettes have been targeted to the ROSA26 locus.
The tamoxifen-inducible Cre system
To allow temporal control of Cre activity, fusion proteins have been constructed between Cre and the ligand-binding domain of steroid hormone receptors (Fig. 1A). The most commonly used variant is a fusion between Cre and a mutated ligand-binding domain of the estrogen receptor (CreERT2) [18-20]. ERT2 binds weakly to endogenous estrogens and strongly to 4-hydroxy tamoxifen (4OH-T), the active metabolite of the synthetic steroid tamoxifen (tmx). Administration of tmx or 4OH-T by itself can be toxic, resulting in various embryonic phenotypes if administered up to about E11.5, or abortion if administered at later stages [21]. Moreover, tmx dosing can cause a transient increase in blood pressure in adult mice [22]. For these reasons it is important to titrate the tmx dose to the minimum required for each experiment. Most investigators dose their animals with tmx, which is converted to 4OH-T in the liver. 4OH-T can also be administered directly, but kinetics of Cre activation and drug metabolism will be different.
The CreERT2 fusion protein is cytoplasmic. Upon binding to tmx, CreERT2 translocates to the nucleus where it accesses the LoxP sites. Earlier CreER and CreERT2 versions can be somewhat leaky when expressed from a strong promoter. However, such strains can still be highly informative if the correct controls are performed [23]. Recombination rates are very sensitive to the levels of CreERT2 expression [24] and to the length of time the protein spends in the nucleus. This is dependent on tmx dose and frequency of dosing. Elegant studies using a cartilage-specific CreERT fusion protein demonstrated that subsequent to a single intraperitoneal injection of tmx into a pregnant female, reporter activity could be detected within 8 hours and that recombination was complete within 24 hours [25]. In addition, it has been reported that administration of tmx to pregnant females via oral gavage, rather than intraperitoneal injection, results in more efficient labeling and less embryonic toxicity [26]. The CreER2T system has been widely used in the lung, although the extent of recombination, and most effective tmx dosing strategy, must be determined empirically for each CreERT2 mouse strain [27, 28]. In addition, the recombination rate between each pair of LoxP sites also varies and must be determined experimentally [29]. Another variant of the CreER system has the ERT fused to both the amino and carboxy terminals of Cre (known as mER-Cre-mER) [30]. The mER-Cre-mER has not been used widely in pulmonary research.
Multiple transgenic strains with widespread expression of tamoxifen-inducible Cre have been generated and are potentially useful in the adult lung for deletion of genes with cell type specific expression. Representatives of these lines are beta-actin-Cre [31] and actin-CreERT [32], which have been used successfully in the developing lung mesenchyme [33], both direct Cre expression in multiple lung cell types. Similarly, a CMV-CreERT mouse line directs expression of CreER in most cell types [19], and has been successfully used to study the role of Sox2 in the adult tracheal epithelium [34]. In addition, four independent RosaCreERT2 strains exist that allow ubiquitous expression of CreERT2 from the ROSA26 locus [29, 35-38].
The split-Cre system
The doxycycline and Cre/CreER systems are both dependent on the availability of a cell type specific promoter to restrict gene expression to the cells of interest. However the variety of cell type specific promoters is limited. The split-Cre system was developed to overcome these limitations [39]. In this system the Cre protein is split into two halves that are expressed from different promoters. Individually these two parts of the protein are inactive. When both promoters are activated in the same cell, inter-molecular complementation occurs and Cre is functional. This system has been successfully applied to the mouse brain to target populations of rare stem cells, which had previously been defined by flow cytometry only [40]. An inducible split CreERT2 system has also been developed and shown to function in vitro [41]. In vivo application of the split Cre in the lung will be beneficial to advance the field of lineage relationships and progenitor cells.
Cre toxicity
Off target effects of the Cre recombinase have been reported in several systems [42], including the lung [43, 44]. These off target effects are probably due to endogenous cryptic LoxP sites within the mammalian genome that cause cytotoxic chromosomal rearrangements when activated [45, 46]. Not every Cre strain shows off-target effects and this variation is likely to result from differences in levels of Cre protein expression. Cre toxicity has led to off-target phenotypes and made interpretation of some experiments difficult. In particular, some Cre lines demonstrate Cre activity in the germline, which results in recombination of the floxed allele independent of the regulatory element driving Cre ([47] and B. R. Stripp personal communication). Such sex-specific effects can usually be avoided by transmitting the Cre via the female or male germline respectively. The use of dox or tmx-dependent Cre strains limits the amount of time Cre spends in the nucleus and decreases Cre toxicity, which also highlights the importance of using the minimum dose of dox or tmx for each experiment. In addition, it is crucial to perform the correct controls: 1) dox and tmx treatment of single transgenic mice, 2) dox and tmx treatment of mice with all transgenes in a non floxed background 3) untreated mice containing all transgenes in the floxed background. Cre mouse strains, often in combination with doxycycline (dox) or tmx, are probably the most widely used animals for manipulating gene expression in the lung.
Flipase
The FLP-FRT system is similar to the Cre-Lox system and is becoming more frequently used in mouse-based research. It involves using Flippase (FLP) recombinase, derived from the yeast Saccharomyces cerevisiae [48]. FLP recognizes a pair of FLP recombinase target (FRT) sequences that flank a genomic region of interest. RosaFLPe is a mouse line with a ubiquitous FLP expression [49]. A useful reporter mouse for this recombination is the Flp indicator mouse expressing alkaline phosphatase from the ROSA26 locus [50]. However, despite many attempts it has been difficult to generate lung specific Flippase mice (A.K. Perl, J. Whitsett unpublished data). Nevertheless, a Flp-inducible allele of K-ras was recently activated in the lung using a lentivirus-encoded FLP protein [51].
The doxycycline system
The tetracycline (tet) inducible system was developed independently to the Cre-LoxP system and has different advantages and limitations. The tet system in vivo consists of two transgenic mouse lines, an activator line and an operator line. The activator line expresses either the tetracycline activator (TA, tet-off) or the reverse tetracycline responsive transactivator (rtTA, tet-on) in a tissue specific manner. The operator line carries a transgene of interest under control of the (tetO)7CMV operator (tetO). In double transgenic mice, doxycycline either causes the tTA to bind the tetO sequence, suppressing transcription (Tet-off), or causes the rtTA to bind to the tetO sequence, activating transcription of the gene of interest (Tet-on) (Fig. 1C) [52, 53]. The major advantage of this system is to reversibly turn genes on and off and study the effects of genes at specific times. In the lung this system was successfully used in 2000 to conditionally activate FGF7 during lung development [54]. The technical aspects of using doxycycline to activate genes were described using a luciferase reporter mouse [55, 56] and the limitations of the system were reported in 2006 [57].
The use of the rtTA system was then further expanded by the combination of the rtTA system with activation of a tetO-Cre transgene [58, 59]. The cell specific, dox dependent Cre expression enables permanent endogenous gene inactivation or transgene activation at any given time during development, or in adult animals (Fig. 1D) [58]. This dox-dependent Cre activation has been shown to be very useful for in vivo cell lineage labeling and conditional deletion of genes that otherwise would result in lethal phenotypes. It has also recently been used to conditionally deplete specific cell populations and study epithelial regeneration [60].
Combining tissue specific Cre lines with Rosa26rtTA transgenic mice results in Cre-mediated rtTA expression (Fig. 1D). These mice have Cre-inducible expression of rtTA and can be used to achieve spatially and temporally controlled transgene expression in a wide variety of settings simply by crossing to any existing mice carrying cell type-specific Cre recombinase and tet-O-regulatable responder genes [61, 62].
Off-target toxicity of rtTA and doxycycline has been reviewed previously [43, 57, 63]. Briefly, these toxicities were unrelated to the effects of the transgene and varied both with specific mouse strain and genetic background. Off-target effects can influence both lung morphogenesis and perinatal survival and most often result in airspace enlargement. In addition, doxycycline is a matrix metalloproteinase inhibitor and has been shown to promote pulmonary hypertension after hypoxia, and to attenuate mucin production [64-66]. More recently it has been shown that tracheal Clara cells are sensitive to doxycycline treatment [44]. Doxycycline is stored in tissues and will be released over time. The timing and duration of treatment needed to target subsets of lung epithelial cells has been described and we highly recommend limiting time and dose of doxycycline exposure [58, 59]. With the new generation rtTA constructs, that are more sensitive to low doses of doxycycline (for example, rtTA2S-M2), off-target effects of doxycycline will become less significant, but dosing regimens will be more important [67, 68]. In all dox experiments, it is vital to perform the correct controls. To control for phenotypes not related to the activation/inactivation of the gene of interest, single transgenic mice, and mice containing all transgenes, should be tested in the absence of doxycycline. Littermate controls should be used where possible to minimize strain or age related variability. Experiments should also be controlled for weight and gender differences.
“Knock-in” versus transgenic mice
There are multiple methods for producing Cre or tTA/rtTA mouse strains. These include generating transgenic animals by pronuclear injection, BAC transgenics (Bacterial Artificial Chromosome) and gene targeting by homologous recombination in ES cells (“knock-ins”). The method used affects the properties of the resulting mouse strain and it is important to be aware of the benefits and limitations of each method.
  • The “transgene” method largely depends on the availability of defined regulatory sequences to drive expression in the cell of interest. When a characterized promoter is available transgenic mice can be generated rapidly by injection of the construct into the pronucleus of a fertilized egg. However, the transgene inserts randomly and expression of the exogenous gene is highly susceptible to the chromosomal environment of the insertion site (heterochromatin versus euchromatin). This phenomenon is known as position-effect variegation and explains why founders with independent insertion sites can have different expression patterns of the transgene. Transgenes can also be silenced over an animal’s lifetime, or sometimes after multiple generations. In addition, the integration of the transgene can cause a mutation in a chromosomal gene [69]. To overcome these problems transgenic constructs can be targeted to a defined locus in ES cells (e.g. Hprt1 [70]) and new techniques for integrating transgenic constructs into defined loci by cassette exchange should make this a very rapid technique [10, 71] [72].
  • BAC (Bacterial Artificial Chromosome) transgenes are large and are injected into the pronucleus of a fertilized egg. For BAC transgenes the Cre or rtTA sequences are usually placed in the ATG of a known gene, which is expressed in the cells of interest. Due to their large size the BAC constructs usually contain most, or all, of the enhancer sequences which normally regulate expression of that gene. The BAC size also makes them less susceptible to positional effects and silencing. However, their size also means that they can include extra copies of flanking genes or regulatory RNAs, which could cause an unrelated phenotype.
  • To generate “knock-ins” Cre or rtTA sequences are usually placed in the ATG of an endogenous chromosomal gene by homologous recombination in ES cells. This simultaneously generates a null allele, which can cause a haplo-insufficiency phenotype. An alternative technique is to target the Cre or rtTA sequences to the 3′ UTR of a locus using an internal ribosome entry site (IRES), or 2A cleavage site, to create a bicistronic mRNA transcript. Since the 3′ UTR can contain important regulatory sequences, these transgenic lines need to be tested for phenotypes and for expression pattern of the transgene. Knock-ins usually carry a single copy of Cre or rtTA, which makes them less-leaky, but also decreases sensitivity compared with the transgenic strains which usually contain multiple copies.
Many of the mice we mention in this review are available form the Jackson Laboratories (http://jaxmice.jax.org). Most importantly the Jackson Laboratories has also developed useful databases. These include, MGI (Mouse Genome Informatics), which provides access to integrated data on mouse genes and genome features, from sequences and genomic maps to gene expression and disease models (http://www.informatics.jax.org/). The IMSR (International Mouse Strain Resource), which is a searchable online database of mouse strains and stocks available worldwide, including inbred, mutant, and genetically engineered mice (http://www.findmice.org/). And the MPD (Mouse Phenome Database), which is a collaborative collection of baseline phenotypic data on inbred mouse strains. The MPD includes data sets, protocols, projects and publications, and SNPs (http://phenome.jax.org/).
1) Epithelial lines
The lung buds from the foregut endoderm around embryonic day (E) 9.5 in mice and continues to develop by branching morphogenesis. By contrast, the trachea and esophagus separate from the foregut just ventral to the lung buds between E10.0 and E11.5 and then increase in length and diameter during development. A number of mouse strains suitable for conditional gene activation in the developing lung have been developed using genes expressed in the foregut endoderm. However, with the exception of Sftpc lines, these are not unique to the developing lung. The choice of line depends partly on the timing of activity required: 1) in the undivided foregut (that is, throughout the entire lung, trachea and esophagus), or 2) more lung-specific. As differentiated cell types appear during lung development they can be targeted with cell type specific mouse lines.
1a) Embryonic epithelial progenitors
Sonic hedgehog (Shh) is expressed in a highly dynamic pattern during embryogenesis. The Shh-Cre line is a knock-in of a GFP-Cre fusion protein [73] and has been shown to activate reporter recombination in the ventral foregut endoderm by E9.5, prior to lung budding and tracheal/esophageal separation [74]. It has successfully been used to study both tracheal and lung development [11, 75, 76].
Islet1 is expressed in many tissues during embryogenesis including the limb and heart, but is enriched in the lung epithelial precursors at E9.5 [77]. The Islet1Cre line is a knock-in strain [78] and drives Cre expression throughout the pharyngeal endoderm, including the pulmonary epithelial precursors, and in a subset of mesenchymal cells [79]. Although it is not epithelial specific, it has been successfully used to study Fgf8 function during lung development [80].
Transgenic mice driving Cre from a 4.8 kb fragment of the Gata5 promoter (Gata5Cre) mediate recombination in the developing epicardium and also throughout the developing lung endoderm [81, 82]. However, onset of recombination and potential activity in the developing trachea has yet to be determined.
The transcription factor Id2 is dynamically expressed in various cell types during embryogenesis and in the adult. The Id2-CreERT2 line is a knock-in of the CreERT2 fusion [83]. In the developing lung this strain displays tamoxifen-dependent Cre-mediated recombination in the distal epithelial tips and, at a lower level, in a sub-set of mesenchymal cells.
Nkx2-5 is one of the earliest cardiac-specific markers in vertebrate embryos. The Nkx2-5 Cre line is a knock-in [84] and drives recombination in the developing proepicardium and subsequently throughout the myocardium and the first pharyngeal arch [85]. Recombination occurs in both the foregut endoderm and surrounding mesoderm prior to E9.5 before lung budding or tracheal-esophogeal separation. Nkx2.5-Cre has successfully been used to inactivate epithelial expression of Sox2 in the developing respiratory tract [34].
Nkx2.1 (also known as Ttf1 and Titf1) is expressed in the domain of the ventral foregut, which will give rise to both the lung and trachea. Nkx2.1 is also expressed in thyroid progenitors and regions of the developing brain [86]. An Nkx2.1-Cre BAC transgenic mouse [87] functions throughout the lung and tracheal epithelium and has been successfully used in a number of studies [88, 89].
Subsequent to budding, the lung epithelium transcribes Sftpc (SpC, or Surfatant associated protein C). Since the human SFTPC promoter is active once endoderm has been committed to becoming lung it is very useful for studying early lung development. The SFTPC lines are discussed below. As lung development proceeds more restricted progenitor cells, such as bronchiolar progenitors, are hypothesized to exist [76]. Tools for manipulating gene expression specifically in such progenitor cells would be highly desirable.
1b) Alveolar type II cells
The 3.7 Kb fragment of the human SFTPC promoter is one of the most widely used promoters to generate constitutive and conditional transgenic mouse strains that target the respiratory epithelium [3]. The advantage of the SFTPC promoter is that it is very lung-specific and off-target effects on other organs are extremely rare. In the adult lung SFTPC promoter activity is restricted to alveolar type II cells and subsets of cuboidal bronchiolar cells. The most widely distributed strains are the SFTPC-rtTA [54, 56, 90] and SFTPC-Cre [91]. The SFTPC-rtTA lines are particularly useful since dox application from E6.5 to E10.5 only targets the progenitor pool of the distal lung epithelium, the parathyroid and the thymus. However, targeting of neuroendocrine cells with these lines was not observed [58]. During organogenesis expression levels of morphogenic genes dynamically change spatially and temporally. With the rtTA line it was possible to activate and inactivate signaling pathways that regulate morphogenic genes in specific compartments at defined times. These studies led to a better understanding of temporal windows for FGF signaling and allowed the process of lung organogenesis to be dissected in greater detail [59, 92, 93]. Most prenatal studies were done using SFTPC-rtTA line 1, which expresses rtTA at high levels, but also shows dox independent gene activation especially around birth and postnatally. Dox independent expression can result in embryonic lethal phenotypes, as it was the case for overexpression of FGF7 and VEGF [54, 90]. To overcome these limitations, a second founder line (SFTPC-rtTA line2) was characterized [90], which expresses lower levels of rtTA and demonstrates less off-dox effects. While line 2 has been demonstrated to work after E14.5 and in adult mice, activation in the developing embryonic endoderm has not been tested. Both lines have been widely distributed in the scientific community.
The SFTPC-Cre transgenic strain contains the human SFTPC promoter fragment, which drives a rabbit β globin intron, followed by Cre recombinase. SFTPC-Cre directs recombination throughout the lung epithelium starting at E10.5 [91]. This strain has also been widely used and has contributed significantly to our understanding of lung development [94, 95]. There are reports of toxic effects on some genetic backgrounds [95, 96]. More recently, it has been demonstrated that the SFTPC-Cre line directs recombination in the male germline, which may have confounded previous studies resulting in the apparent toxicity (B.R. Stripp, personal communication). As long as this transgene is transmitted through the female germline it is very useful to study embryonic lung development.
The Sftpc-CreERT2-rtTA is a knock-in of both CreERT2 and rtTA cassettes just after the stop codon of the endogenous Sftpc gene [97]. This very flexible strain drives both CreERT2 and rtTA expression in mature adult type II cells. It has already proved to be useful for lineage-tracing experiments and will undoubtedly also be widely used for manipulating gene expression.
Other SFTPC-lines are summarized in Table 1. There are alternative promoters (e.g. ABCA3, C/EBPα), which could be potentially used to target alveolar type II cells. However, these promoters will not be lung specific.
Table 1
Table 1
Embryonic epithelial progenitors
1c) Alveolar type I cells
Aquaporin 5 (Aqp5), is expressed predominantly in salivary and lacrimal glands, cornea, trachea, and distal lung [98, 99]. In rat and human lungs, Aqp5 is specifically expressed in alveolar type I (AT1) cells and not in alveolar type II (AT2) cells. In mice Aqp5 expression has also been found in AT2 cells [100]. A Cre-IRES-DsRed cassette has been inserted into exon 1 of the endogenous Aqp5 locus generating the Aqp5-Cre-IRES-DsRed, or ACID mouse [101]. Analysis with the ROSA-mT/mG reporter [102], demonstrated that recombination had occurred in a very high fraction of AT1 cells in the distal lung and not in AT2 cells. However, AT2 recombination in other genetic backgrounds cannot yet be ruled out. This is the first transgenic mouse engineered to express Cre in AT1 cells and it should be very useful for studies of AT1 turnover and function.
Podopladin, or T1 alpha, a gene with unclear function, is expressed in mouse AT1 cells and lymphatics [103]. By contrast, the rat podopladin gene is specific for AT1 cells. A modified rat BAC containing internal ribosome entry site (IRES)-green fluorescent protein (GFP) in the podoplanin 3′UTR has been generated (RTIbac) [104]. RTIbac-transgenic mice expressed rat podoplanin in AT1 cells and in the brain, and expression in AT2 cells, airways, and vascular endothelium was not detected. Modifications of this BAC to express the rtTA or Cre recombinase could make this construct useful for targeting ATI cells.
1d) Bronchiolar Clara Cells
Secretoglobin1a1 (Scgb1a1, also known as CCSP, CC10 and CCA) is expressed in all bronchiolar Clara cells, and at lower levels in most tracheal Clara-like cells. In the rat Scgb1a1 is also expressed in AT2 cells. A rat Scgb1a1 promoter fragment has been used for making various transgenic lines.
A 2.3Kb fragment of the rat Scgb1a1 promoter is sufficient to direct expression in mouse Clara cells [1]. This promoter was subsequently used to generate two independent Scgb1a1-rtTA mouse lines. The first line [56] has been widely distributed within the research community and used successfully in multiple studies. Lineage-tracing showed that this strain has efficient activity in many bronchiolar Clara cells and also a sub-set of AT2 cells [59]. The Scgb1a1-rtTA line 2 targets most Clara cells, but retains little AT2 cell activity [43, 60]. Scgb1a1 is also active in the uterus. However, using luciferase reporter mice no luciferase activity was detected in whole uterus homogenates [56].
The rat Scgb1a1 promoter has also been used to generate a Scgb1a1-rtTA2S-M2 which uses the newer more-sensitive version of rtTA [68]. This strain is reported to have no basal activity and increased doxycycline sensitivity. However, it also has some activity in AT2 cells.
An Scgb1a1-Cre transgenic strain was generated using the rat promoter fragment inserted upstream of the coding sequence for Cre [105, 106]. Lineage tracing shows that this strain directs recombination in the bronchiolar cells, but not in any alveolar cells [107], demonstrating that the insertion site of the transgene has a strong effect on expression pattern.
An Scgb1a1-CreER™ “knock-in” mouse strain was generated by inserting an IRES-CreER™ cassette into the 3′ UTR of the endogenous mouse Scgb1a1 locus [23]. This line was used for detailed lineage-tracing studies, which revealed that it provides specific, tamoxifen dose-dependent Cre activity in up to 90% of bronchiolar Clara cells, and up to 7% of AT2 cells. However, it also displays some tamoxifen-independent activity.
A knock-in Tgfb3-Cre line [108] has recently been used to manipulate Notch signaling in the postnatal lung airways [109]. Reporter analysis suggested that this strain targets the majority (~90%) of Clara cells.
There is evidence to suggest that not all Clara cells are functionally equivalent [110, 111]. Transgenic strains, which target specific sub-sets of Clara cells would be highly desirable for the lung research community.
1e) Ciliated cells
Foxj1 is a transcription factor which is expressed in all multiciliated cells including those of the lung, oviducts, ependyma and testes [112], and various cells with motile cilia [113-115]. A 1Kb fragment of the human FOXJ1 promoter was shown to be sufficient to direct reporter gene expression specifically in all of these cell types in adult mice [116]. This promoter was subsequently used to generate FOXJ1-Cre [117], and FOXJ1-CreERT2 transgenic mice [28]. Both lines drive efficient recombination in ciliated cells of the respiratory tract and have been useful for gene knock-out and lineage-tracing studies. In particular, the FOXJ1-CreERT2 mice were used to determine the average half-life of ciliated cells in the mouse airways [118]. The FOXJ1-CreERT2 strain also unexpectedly directs recombination very efficiently in pericytes (J.R. Rock, B.L.M. Hogan, personal communication). This may reflect a low level of endogenous pericyte Foxj1 expression, or be due to the insertion site of the transgene. Similarly, a recent paper has shown that the same human FOXJ1 promoter can drive expression in human ciliated cells, but also some basal cells, growing at an air-liquid interface [119]. Basal cell expression was not observed in the transgenic animals. However, any future transgenic strains generated with this promoter should be screened for basal cell and pericyte activity.
1f) Basal cells
Published gene expression data have so far not identified a gene that is expressed exclusively in airway basal cells [27]. A split-Cre, or viral, approach may be necessary for airway-specific basal cell genetic manipulation.
Keratin 5 (Krt5) and Keratin 14 (Krt14) promoters have been used to target basal cells in the airway epithelium. All mouse and human airway epithelial basal cells express Krt5 [120, 121]. A human 6kb KRT5 promoter fragment was cloned by the Fuchs lab and successfully used to target epidermal basal cells [122, 123]. Using the same promoter fragment KRT5-CreER2T transgenic mice were generated and used for cell lineage tracing in the airways. These studies demonstrated that airway basal cells are stem cells [27]. This strain has subsequently been used for studying the control of basal cell function [124] and should prove to be generally useful for manipulating gene expression in tracheal basal cells. However, the KRT5-CreER2T transgenic strain is limited as it directs recombination in only about 15% of basal cells in the adult trachea. Moreover, the high levels of transgene activity in the skin and oral epithelium makes this strain extremely difficult to use for studies of oncogenes, as the mice will develop skin and oral tumors before the trachea is affected.
Krt14 is expressed in roughly 30% of mouse tracheal basal cells [120, 121, 125]. Transgenic mice containing the human KRT14 promoter linked to CreERT were generated for use in the skin [126]. In the trachea these KRT14-CreERT transgenic mice allow tmx-induced recombination in an extremely small population of basal cells at steady-state [125]. Following naphthalene injury, most surviving basal cells upregulate Krt14 and also express the transgene [127]. This mouse has not yet been used to manipulate gene expression in airway basal cells. However, a K14-rtTA mouse [128] has been used, in combination with the tetO-Cre strain [58], to direct gene expression in the trachea [44]. These data show the importance of careful control experiments, as in the K14-rtTA strain tracheal Clara cells were sensitive to dox exposure.
1g) Neuroendocrine cells
Pulmonary neuroendocrine (NE) cell differentiation depends on genes which are conserved in the nervous system of many organisms, for example Ascl1, NeuroD, Rb, and Gfi1 [129-132]. A rat NE cell specific promoter has been identified [133] and recently used in an adenovirus to direct gene expression specifically to NE cells [134]. While no transgenic mouse strains, that specifically target NE cells have been generated, such strains could be very useful for studying NE cell function, or their putative role as an airway epithelial stem cell niche [135].
2) Mesenchymal lines
There is still much disagreement over the numbers of different lung mesenchymal cell types and their best markers [136]. The challenge of the available mesenchymal mouse strains is that they are not lung-specific and that many of their expression patterns are highly variable depending on the integration of the transgene. Pod1 (also known as Tcf21) is highly expressed in the mesenchyme of the developing lung, kidney, heart and intestine and may be a useful promoter for more restricted mesenchymal gene manipulation [137]. Tbx genes may also provide useful mesenchymal promoters. Tbx2-5 are expressed in the developing lung mesenchyme [138] and are often used as reporters of embryonic mesenchymal fate [139, 140]. However, the expression patterns of Pod1 and the Tbx genes in the adult lung mesenchyme have yet to be determined.
Dermo1-Cre (also known as Twist2-Cre) is a knock-in of Cre [141] that displays robust recombination in mesenchymal and mesothelial lineages in the lung [142]. It has been widely used for manipulating gene expression in the developing lung mesenchyme [143, 144].
Mesp1-Cre is a knock-in of Cre, replacing the Mesp1 coding region, and drives expression throughout the anterior mesoderm from early gastrulation [145]. It has been successfully used to manipulate gene expression in the developing lung mesoderm [80].
Fibroblast specific protein 1 (FSP1), also known as S100A4, has been reported as a fibroblast specific gene, but is also induced in epithelial cells during injury and tumor progression [146, 147]. The FSP1 promoter has been used to generate various FSP1-Cre mice with variable success [148]. The use of these mice to study mesenchymal cells in the lung remains controversial.
Adipocyte lipid-binding protein 2 (aP2, also known as fatty acid binding protein 4, Fabp4) is expressed in alveolar type II cells and interstitial lipofibroblasts. The mouse aP2 promoter was used to generate aP2-Cre and aP2-CreERT2 mice [149]. While this aP2-Cre line does not target AT2 cells it does target a subset of alveolar fibroblasts. Induction of recombination with the aP2-CreERT2 in adult mice was not observed (A.K. Perl, unpublished data).
The SM22-alpha (SM22α or transgelin) promoter was used to generate SM22-rtTA mice. This line provides a “Tet-On” tool that allows the inducible expression of genes in smooth muscle cells [150]. Expression is mainly in the vascular smooth muscle and the SM22 promoter was used to generate several transgenic and knock-in CreERT2-expressing lines with varying expression patterns: with the highest levels detected in the aorta, intestine and uterus. However, none of these lines is particularly efficient, even in the vascular smooth muscle [151].
SMA (Smooth Muscle alpha Actin, or Acta2) is expressed in all smooth muscle cells in the adult, and also transiently in myocardiocytes and skeletal muscle during embryonic development [152, 153]. In lung parenchyma SMA is expressed during alveolarization, realveolarization, and during the development of lung fibrosis after bleomycin or hyperoxic injury [154-157]. Mice with a murine αSMA-Cre transgene express Cre in the airway smooth muscle and lung vasculature [158]. This line is not inducible which limits postnatal studies. More recently an αSMA-CreERT2 BAC transgenic line has been generated and shown to exhibit tamoxifen-dependent Cre activity in all adult smooth muscle, including the lung airways and vasculature [159]. These mice have off target Cre activity only in a small number of cardiomyocytes and should be very useful for future lung studies.
SMMHC (Smooth Muscle Myosin Heavy Chain, or Myh11) is expressed in all smooth muscle cells. Two independent mouse strains expressing Cre from a fragment of the mouse SMMHC promoter have been generated. The expression of the transgene is somewhat variable [160, 161]. This promoter leads to spurious CRE activity in some tissues due to expression in male and female germline [47]. By contrast, a SMMHC-CreERT2 BAC transgenic strain shows inducible Cre activity in all smooth muscle, including the airway smooth muscle and lung vasculature [22]. This strain should be useful for manipulation of gene expression in perivascular and peribronchiolar smooth muscle in the postnatal lung.
3) Mesothelium
To direct Cre expression to the mesothelium of internal organs including the liver, gut and lung a Wt1-Cre YAC (yeast artificial chromosome) transgenic strain was generated [162], This has been shown to be active throughout the lung mesothelium from early developmental stages [163, 164]. However, it may be expressed at low levels in mesenchymal lineage of the embryonic lung and needs to be used with caution (B.L.M. Hogan personal communication). An inducible version would be useful for adult studies.
4) Endothelial lines
Multiple independent transgenic strains express Cre recombinase under the control of the mouse Tek promoter (Endothelial-Specific Receptor Tyrosine Kinase, also known as Tie2), Some of these have been very widely used [165, 166] [167, 168]. In these strains, reporter gene activity was detected in most endothelial cells and blood islands of the extra embryonic mesoderm by E7.5, in the dorsal aorta by E8.5 and in all blood vessels and some blood cells examined at E11.5, indicating that Cre was active in early vascular progenitors, endothelial cells and some hematopoietic cells. This promoter leads to spurious Cre activity in some tissues due to expression in male and female germline [47].
5) Reporters of signaling pathway activity
Wnt pathway
Wnt signaling pathways play divergent roles during development, homeostasis and repair and play a major role in stem cell proliferation and differentiation. Three transgenic reporter lines for Wnt pathway activity have been generated 1), TOPGAL, which reports epithelial Wnt signaling [169], 2) BATGAL with sporadic epithelial and mesenchymal activity [170] and 3) Axin2-lacZ, which is useful to study proximal lung and mesenchymal Wnt signaling [171]. A recent study compared these lines during development and after naphthalene injury [172]. A new reporter line, TCF/Lef:H2B-GFP, has not yet been tested in the lung [173].
Notch pathway
Signals through the Notch receptors are used throughout embryonic development and in the adult to control cellular fate choices. CP-EGFP (also known as TNR) transgenic mice have a transgenic Notch reporter with an enhanced green fluorescent protein (EGFP) placed under the control of 4 tandem copies of a CBF1 (also known as Rbpj) responsive element (4 CBF1 binding site consensus sequences and the basal SV40 promoter) [174, 175]. This strain has been shown to faithfully report Notch activity in the adult trachea [176]. N1IP::CRELOW and N1IP::CREHI are knock-ins of Cre, replacing the Notch1 coding region (Notch1 Intramembrane Proteolysis) and allow lineage studies of descendents of cells after Notch 1 activation [164, 177]. However, this Cre line identifies each cell lineage, which has previously experienced Notch activity and does not report on current signaling events. Comparison of various Notch reporter lines will shed better light on cell fate decisions and lineage relationships and lead to a better understanding of stem cell biology and interactions of epithelium, mesenchyme, mesothelium and endothelium during development and repair.
6) Virus-mediated transient lung transgenics
The lung is exposed to the external environment and multiple groups have taken advantage of this by administering viruses to manipulate gene expression in the adult mouse lung epithelium. The most widespread system is intranasal administration of an adenovirus expressing Cre from a ubiquitous CMV promoter (AdenoCre) to activate the expression of oncogenes and model lung cancer [178]. A similar adenovirus-based approach has been taken to transiently overexpress specific genes throughout the lung epithelium (for example, [179]). More recently, adenoviruses using Scgb1a1, rat SftpC, or rat CGRP promoter fragments to direct Cre expression to specific epithelial cell types have been developed [134]. Lentiviral vectors containing specific promoters for manipulating gene expression in restricted adult lung epithelial cell types have also been reported [180, 181]. In addition, Adeno-Associated Virus (AAV) transduction of mouse lung epithelial progenitors has also been demonstrated [182]. The use of viral systems is likely to become more widespread over the next few years, particularly for epithelial studies.
7) Conclusions/outlooks
Transgenic mice have been instrumental in developing our current understanding of lung embryonic development, adult homeostasis and repair. However, it is important to remember that all transgenic approaches have limitations, which can only be overcome by integrating findings from different lines and performing all the appropriate controls. New developments in mouse conditional genetics have the potential to further enhance our understanding of lung development and disease. Moreover, optimizing mouse strains of the existing doxycycline and Cre systems will increase flexibility and improve experimental design. For example, using the newer more dox-sensitive rtTA gene activation (rtTA2S-M2), or extremely low doses of tamoxifen in CreER based transgenic mice, will allow recombination in single cells and enable clonal cell type-specific gene manipulation. Due to the lack of lung-specific mesenchymal and endothelial gene expression, more lines need to be characterized for their usefulness in targeting specific subsets of mesenchymal and endothelial cells. On the other hand, complex targeting systems, such as the split-Cre will be helpful to target subsets of epithelial progenitor cells or specific mesenchymal cell lineages. Recently the applicability of the Flipase system to the lung has been demonstrated by combining Cre and FLP to independently control recombination of p53 and kRas in lung tumor progression [51]. Development of tools based on flippase and viruses will further expand the combinatorial use of the existing mouse lines and help to develop newer lines, possibly overcoming the problems of off-target activation, lack of cell type specificity and lack of adult regulation. In addition, the generation of new publicly-available floxed alleles by the International Mouse Knock out Consortium (http://www.knockoutmouse.org/) and the use of transgenic mice expressing conditional RNA interference constructs, should facilitate mouse conditional genetic analysis.
Table 2
Table 2
Mature epithelium
Table 3
Table 3
Mesenchymal and Endothelial Lines
Table 4
Table 4
Reporter lines and others
Acknowledgements
Jeffrey Whitsett, Jason Rock, Barry Stripp and Brigid Hogan very kindly shared unpublished data. In addition, we thank Jeffrey Whitsett, Brigid Hogan, Jim Bridges, and members of our laboratories for critical comments on the manuscript.
1. Stripp BR, et al. cis-acting elements that confer lung epithelial cell expression of the CC10 gene. J Biol Chem. 1992;267(21):14703–12. [PubMed]
2. Stripp BR, Huffman JA, Bohinski RJ. Structure and regulation of the murine Clara cell secretory protein gene. Genomics. 1994;20(1):27–35. [PubMed]
3. Wert SE, et al. Transcriptional elements from the human SP-C gene direct expression in the primordial respiratory epithelium of transgenic mice. Dev Biol. 1993;156(2):426–43. [PubMed]
4. Sternberg N, Hamilton D. Bacteriophage P1 site-specific recombination. I. Recombination between loxP sites. J Mol Biol. 1981;150(4):467–86. [PubMed]
5. Sauer B, Henderson N. Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc Natl Acad Sci U S A. 1988;85(14):5166–70. [PubMed]
6. Sauer B. Inducible gene targeting in mice using the Cre/lox system. Methods. 1998;14(4):381–92. [PubMed]
7. Nagy A. Cre recombinase: the universal reagent for genome tailoring. Genesis. 2000;26(2):99–109. [PubMed]
8. Livet J, et al. Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature. 2007;450(7166):56–62. [PubMed]
9. Snippert HJ, et al. Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Cell. 2010;143(1):134–44. [PubMed]
10. Burlison JS, et al. Pdx-1 and Ptf1a concurrently determine fate specification of pancreatic multipotent progenitor cells. Dev Biol. 2008;316(1):74–86. [PMC free article] [PubMed]
11. Domyan ET, Sun X. Patterning and plasticity in development of the respiratory lineage. Dev Dyn. 2011;240(3):477–85. [PMC free article] [PubMed]
12. Lu Y, et al. Evidence that SOX2 overexpression is oncogenic in the lung. PLoS One. 2010;5(6):e11022. [PMC free article] [PubMed]
13. Lobe CG, et al. Z/AP, a double reporter for cre-mediated recombination. Dev Biol. 1999;208(2):281–92. [PubMed]
14. Novak A, et al. Z/EG, a double reporter mouse line that expresses enhanced green fluorescent protein upon Cre-mediated excision. Genesis. 2000;28(3-4):147–55. [PubMed]
15. Friedrich G, Soriano P. Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice. Genes Dev. 1991;5(9):1513–23. [PubMed]
16. Zambrowicz BP, et al. Disruption of overlapping transcripts in the ROSA beta geo 26 gene trap strain leads to widespread expression of beta-galactosidase in mouse embryos and hematopoietic cells. Proc Natl Acad Sci U S A. 1997;94(8):3789–94. [PubMed]
17. Soriano P. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet. 1999;21(1):70–1. [PubMed]
18. Indra AK, et al. Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: comparison of the recombinase activity of the tamoxifen-inducible Cre-ER(T) and Cre-ER(T2) recombinases. Nucleic Acids Res. 1999;27(22):4324–7. [PMC free article] [PubMed]
19. Feil R, et al. Ligand-activated site-specific recombination in mice. Proc Natl Acad Sci U S A. 1996;93(20):10887–90. [PubMed]
20. Feil R, et al. Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains. Biochem Biophys Res Commun. 1997;237(3):752–7. [PubMed]
21. Hayashi S, McMahon AP. Efficient recombination in diverse tissues by a tamoxifen-inducible form of Cre: a tool for temporally regulated gene activation/inactivation in the mouse. Dev Biol. 2002;244(2):305–18. [PubMed]
22. Wirth A, et al. G12-G13-LARG-mediated signaling in vascular smooth muscle is required for salt-induced hypertension. Nat Med. 2008;14(1):64–8. [PubMed]
23. Rawlins EL, et al. The role of Scgb1a1+ Clara cells in the long-term maintenance and repair of lung airway, but not alveolar, epithelium. Cell Stem Cell. 2009;4(6):525–34. [PMC free article] [PubMed]
24. Buelow B, Scharenberg AM. Characterization of parameters required for effective use of tamoxifen-regulated recombination. PLoS One. 2008;3(9):e3264. [PMC free article] [PubMed]
25. Nakamura E, Nguyen MT, Mackem S. Kinetics of tamoxifen-regulated Cre activity in mice using a cartilage-specific CreER(T) to assay temporal activity windows along the proximodistal limb skeleton. Dev Dyn. 2006;235(9):2603–12. [PubMed]
26. Park EJ, et al. System for tamoxifen-inducible expression of cre-recombinase from the Foxa2 locus in mice. Dev Dyn. 2008;237(2):447–53. [PubMed]
27. Rock JR, et al. Basal cells as stem cells of the mouse trachea and human airway epithelium. Proc Natl Acad Sci U S A. 2009;106(31):12771–5. [PubMed]
28. Rawlins EL, et al. Lung development and repair: contribution of the ciliated lineage. Proc Natl Acad Sci U S A. 2007;104(2):410–7. [PubMed]
29. Vooijs M, Jonkers J, Berns A. A highly efficient ligand-regulated Cre recombinase mouse line shows that LoxP recombination is position dependent. EMBO Rep. 2001;2(4):292–7. [PubMed]
30. Zhang Y, et al. Inducible site-directed recombination in mouse embryonic stem cells. Nucleic Acids Res. 1996;24(4):543–8. [PMC free article] [PubMed]
31. Ma W, et al. Hepatic vascular tumors, angiectasis in multiple organs, and impaired spermatogenesis in mice with conditional inactivation of the VHL gene. Cancer Res. 2003;63(17):5320–8. [PubMed]
32. Guo C, Yang W, Lobe CG. A Cre recombinase transgene with mosaic, widespread tamoxifen-inducible action. Genesis. 2002;32(1):8–18. [PubMed]
33. White AC, Lavine KJ, Ornitz DM. FGF9 and SHH regulate mesenchymal Vegfa expression and development of the pulmonary capillary network. Development. 2007;134(20):3743–52. [PMC free article] [PubMed]
34. Que J, et al. Multiple roles for Sox2 in the developing and adult mouse trachea. Development. 2009;136(11):1899–907. [PubMed]
35. Takeda K, Cowan A, Fong GH. Essential role for prolyl hydroxylase domain protein 2 in oxygen homeostasis of the adult vascular system. Circulation. 2007;116(7):774–81. [PubMed]
36. Cheng Y, et al. The Engrailed homeobox genes determine the different foliation patterns in the vermis and hemispheres of the mammalian cerebellum. Development. 2010;137(3):519–29. [PubMed]
37. de Luca C, et al. Complete rescue of obesity, diabetes, and infertility in db/db mice by neuron-specific LEPR-B transgenes. J Clin Invest. 2005;115(12):3484–93. [PMC free article] [PubMed]
38. Ventura A, et al. Restoration of p53 function leads to tumour regression in vivo. Nature. 2007;445(7128):661–5. [PubMed]
39. Hirrlinger J, et al. Split-CreERT2: temporal control of DNA recombination mediated by split-Cre protein fragment complementation. PLoS One. 2009;4(12):e8354. [PMC free article] [PubMed]
40. Beckervordersandforth R, et al. In vivo fate mapping and expression analysis reveals molecular hallmarks of prospectively isolated adult neural stem cells. Cell Stem Cell. 2010;7(6):744–58. [PubMed]
41. Hirrlinger J, et al. Split-cre complementation indicates coincident activity of different genes in vivo. PLoS One. 2009;4(1):e4286. [PMC free article] [PubMed]
42. Naiche LA, Papaioannou VE. Cre activity causes widespread apoptosis and lethal anemia during embryonic development. Genesis. 2007;45(12):768–75. [PubMed]
43. Perl AK, Zhang L, Whitsett JA. Conditional expression of genes in the respiratory epithelium in transgenic mice: cautionary notes and toward building a better mouse trap. Am J Respir Cell Mol Biol. 2009;40(1):1–3. [PMC free article] [PubMed]
44. Smith RW, Hicks DA, Reynolds SD. ROLES FOR {beta}-CATENIN AND DOXYCYCLINE IN REGULATION OF RESPIRATORY EPITHELIAL CELL FREQUENCY AND FUNCTION. Am. J. Respir. Cell Mol. Biol. 2011:2011–0099OC. [PMC free article] [PubMed]
45. Schmidt EE, et al. Illegitimate Cre-dependent chromosome rearrangements in transgenic mouse spermatids. Proc Natl Acad Sci U S A. 2000;97(25):13702–7. [PubMed]
46. Semprini S, et al. Cryptic loxP sites in mammalian genomes: genome-wide distribution and relevance for the efficiency of BAC/PAC recombineering techniques. Nucleic Acids Res. 2007;35(5):1402–10. [PMC free article] [PubMed]
47. de Lange WJ, et al. Germ line activation of the Tie2 and SMMHC promoters causes noncell-specific deletion of floxed alleles. Physiol Genomics. 2008;35(1):1–4. [PMC free article] [PubMed]
48. Sadowski PD. The Flp recombinase of the 2-microns plasmid of Saccharomyces cerevisiae. Prog Nucleic Acid Res Mol Biol. 1995;51:53–91. [PubMed]
49. Farley FW, et al. Widespread recombinase expression using FLPeR (flipper) mice. Genesis. 2000;28(3-4):106–10. [PubMed]
50. Awatramani R, et al. An Flp indicator mouse expressing alkaline phosphatase from the ROSA26 locus. Nat Genet. 2001;29(3):257–9. [PubMed]
51. Young NP, Crowley D, Jacks T. Uncoupling cancer mutations reveals critical timing of p53 loss in sarcomagenesis. Cancer Res. 2011;71(11):4040–7. [PMC free article] [PubMed]
52. Gossen M, Bujard H. Efficacy of tetracycline-controlled gene expression is influenced by cell type: commentary. Biotechniques. 1995;19(2):213–6. discussion 216-7. [PubMed]
53. Kistner A, et al. Doxycycline-mediated quantitative and tissue-specific control of gene expression in transgenic mice. Proc Natl Acad Sci U S A. 1996;93(20):10933–8. [PubMed]
54. Tichelaar JW, Lu W, Whitsett JA. Conditional expression of fibroblast growth factor-7 in the developing and mature lung. J Biol Chem. 2000;275(16):11858–64. [PubMed]
55. Schultze N, et al. Efficient control of gene expression by single step integration of the tetracycline system in transgenic mice. Nat Biotechnol. 1996;14(4):499–503. [PubMed]
56. Perl AK, Tichelaar JW, Whitsett JA. Conditional gene expression in the respiratory epithelium of the mouse. Transgenic Res. 2002;11(1):21–9. [PubMed]
57. Whitsett JA, Perl AK. Conditional control of gene expression in the respiratory epithelium: A cautionary note. Am J Respir Cell Mol Biol. 2006;34(5):519–20. [PubMed]
58. Perl AK, et al. Early restriction of peripheral and proximal cell lineages during formation of the lung. Proc Natl Acad Sci U S A. 2002;99(16):10482–7. [PubMed]
59. Perl AK, et al. Conditional recombination reveals distinct subsets of epithelial cells in trachea, bronchi, and alveoli. Am J Respir Cell Mol Biol. 2005;33(5):455–62. [PMC free article] [PubMed]
60. Perl AK, Riethmacher D, Whitsett JA. Conditional depletion of airway progenitor cells induces peribronchiolar fibrosis. Am J Respir Crit Care Med. 2011;183(4):511–21. [PMC free article] [PubMed]
61. Belteki G, et al. Conditional and inducible transgene expression in mice through the combinatorial use of Cre-mediated recombination and tetracycline induction. Nucleic Acids Res. 2005;33(5):e51. [PMC free article] [PubMed]
62. Hochedlinger K, et al. Ectopic expression of Oct-4 blocks progenitor-cell differentiation and causes dysplasia in epithelial tissues. Cell. 2005;121(3):465–77. [PubMed]
63. Morimoto M, Kopan R. rtTA toxicity limits the usefulness of the SP-C-rtTA transgenic mouse. Dev Biol. 2009;325(1):171–8. [PMC free article] [PubMed]
64. Vieillard-Baron A, et al. Inhibition of matrix metalloproteinases by lung TIMP-1 gene transfer or doxycycline aggravates pulmonary hypertension in rats. Circ Res. 2000;87(5):418–25. [PubMed]
65. Ren S, et al. Doxycycline attenuates acrolein-induced mucin production, in part by inhibiting MMP-9. Eur J Pharmacol. 2011;650(1):418–23. [PubMed]
66. Ohbayashi H. Matrix metalloproteinases in lung diseases. Curr Protein Pept Sci. 2002;3(4):409–21. [PubMed]
67. Urlinger S, et al. Exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity. Proc Natl Acad Sci U S A. 2000;97(14):7963–8. [PubMed]
68. Duerr J, et al. Use of a New Generation Reverse Tetracycline Transactivator System for Quantitative Control of Conditional Gene Expression in the Murine Lung. Am J Respir Cell Mol Biol. 2010 [PubMed]
69. Martin DI, Whitelaw E. The vagaries of variegating transgenes. Bioessays. 1996;18(11):919–23. [PubMed]
70. Yang GS, et al. Next generation tools for high-throughput promoter and expression analysis employing single-copy knock-ins at the Hprt1 locus. Genomics. 2009;93(3):196–204. [PubMed]
71. Premsrirut PK, et al. A rapid and scalable system for studying gene function in mice using conditional RNA interference. Cell. 2011;145(1):145–58. [PMC free article] [PubMed]
72. Chen SX, et al. Quantification of factors influencing fluorescent protein expression using RMCE to generate an allelic series in the ROSA26 locus in mice. Dis Model Mech. 2011;4(4):537–47. [PMC free article] [PubMed]
73. Harfe BD, et al. Evidence for an expansion-based temporal Shh gradient in specifying vertebrate digit identities. Cell. 2004;118(4):517–28. [PubMed]
74. Harris KS, et al. Dicer function is essential for lung epithelium morphogenesis. Proc Natl Acad Sci U S A. 2006;103(7):2208–13. [PubMed]
75. Harris-Johnson KS, et al. beta-Catenin promotes respiratory progenitor identity in mouse foregut. Proc Natl Acad Sci U S A. 2009;106(38):16287–92. [PubMed]
76. Tsao PN, et al. Notch signaling controls the balance of ciliated and secretory cell fates in developing airways. Development. 2009;136(13):2297–307. [PubMed]
77. Millien G, et al. Characterization of the mid-foregut transcriptome identifies genes regulated during lung bud induction. Gene Expr Patterns. 2008;8(2):124–39. [PMC free article] [PubMed]
78. Yang L, et al. Isl1Cre reveals a common Bmp pathway in heart and limb development. Development. 2006;133(8):1575–85. [PubMed]
79. Park KS, et al. Sox17 influences the differentiation of respiratory epithelial cells. Dev Biol. 2006;294(1):192–202. [PubMed]
80. Yu S, et al. Fetal and postnatal lung defects reveal a novel and required role for Fgf8 in lung development. Dev Biol. 2010;347(1):92–108. [PubMed]
81. Merki E, et al. Epicardial retinoid X receptor alpha is required for myocardial growth and coronary artery formation. Proc Natl Acad Sci U S A. 2005;102(51):18455–60. [PubMed]
82. Xing Y, et al. Signaling via Alk5 controls the ontogeny of lung Clara cells. Development. 2010;137(5):825–33. [PubMed]
83. Rawlins EL, et al. The Id2+ distal tip lung epithelium contains individual multipotent embryonic progenitor cells. Development. 2009;136(22):3741–5. [PubMed]
84. Moses KA, et al. Embryonic expression of an Nkx2-5/Cre gene using ROSA26 reporter mice. Genesis. 2001;31(4):176–80. [PubMed]
85. Zhou B, et al. Epicardial progenitors contribute to the cardiomyocyte lineage in the developing heart. Nature. 2008;454(7200):109–13. [PMC free article] [PubMed]
86. Lazzaro D, et al. The transcription factor TTF-1 is expressed at the onset of thyroid and lung morphogenesis and in restricted regions of the foetal brain. Development. 1991;113(4):1093–104. [PubMed]
87. Xu Q, Tam M, Anderson SA. Fate mapping Nkx2.1-lineage cells in the mouse telencephalon. J Comp Neurol. 2008;506(1):16–29. [PubMed]
88. Li C, et al. Stabilized beta-catenin in lung epithelial cells changes cell fate and leads to tracheal and bronchial polyposis. Dev Biol. 2009;334(1):97–108. [PMC free article] [PubMed]
89. Tiozzo C, et al. Deletion of Pten expands lung epithelial progenitor pools and confers resistance to airway injury. Am J Respir Crit Care Med. 2009;180(8):701–12. [PMC free article] [PubMed]
90. Akeson AL, et al. Temporal and spatial regulation of VEGF-A controls vascular patterning in the embryonic lung. Dev Biol. 2003;264(2):443–55. [PubMed]
91. Okubo T, et al. Nmyc plays an essential role during lung development as a dosage-sensitive regulator of progenitor cell proliferation and differentiation. Development. 2005;132(6):1363–74. [PubMed]
92. Perl AK, et al. Temporal effects of Sprouty on lung morphogenesis. Dev Biol. 2003;258(1):154–68. [PubMed]
93. Hokuto I, Perl AK, Whitsett JA. Prenatal, but not postnatal, inhibition of fibroblast growth factor receptor signaling causes emphysema. J Biol Chem. 2003;278(1):415–21. [PubMed]
94. Guseh JS, et al. Notch signaling promotes airway mucous metaplasia and inhibits alveolar development. Development. 2009;136(10):1751–9. [PubMed]
95. Eblaghie MC, et al. Evidence that autocrine signaling through Bmpr1a regulates the proliferation, survival and morphogenetic behavior of distal lung epithelial cells. Dev Biol. 2006;291(1):67–82. [PubMed]
96. Jeannotte L, et al. Unsuspected effects of a lung-specific cre deleter mouse line. Genesis. 2011;49(3):152–9. [PubMed]
97. Chapman HA, et al. Integrin alpha6beta4 identifies an adult distal lung epithelial population with regenerative potential in mice. J Clin Invest. 2011 [PMC free article] [PubMed]
98. Funaki H, et al. Localization and expression of AQP5 in cornea, serous salivary glands, and pulmonary epithelial cells. Am J Physiol. 1998;275(4 Pt 1):C1151–7. [PubMed]
99. Raina S, et al. Molecular cloning and characterization of an aquaporin cDNA from salivary, lacrimal, and respiratory tissues. J Biol Chem. 1995;270(4):1908–12. [PubMed]
100. Krane CM, et al. Aquaporin 5-deficient mouse lungs are hyperresponsive to cholinergic stimulation. Proc Natl Acad Sci U S A. 2001;98(24):14114–9. [PubMed]
101. Flodby P, et al. Directed expression of Cre in alveolar epithelial type 1 cells. Am J Respir Cell Mol Biol. 2010;43(2):173–8. [PMC free article] [PubMed]
102. Muzumdar MD, et al. A global double-fluorescent Cre reporter mouse. Genesis. 2007;45(9):593–605. [PubMed]
103. Farr AG, et al. Characterization and cloning of a novel glycoprotein expressed by stromal cells in T-dependent areas of peripheral lymphoid tissues. J Exp Med. 1992;176(5):1477–82. [PMC free article] [PubMed]
104. Vanderbilt JN, et al. Directed expression of transgenes to alveolar type I cells in the mouse. Am J Respir Cell Mol Biol. 2008;39(3):253–62. [PMC free article] [PubMed]
105. Ji H, et al. K-ras activation generates an inflammatory response in lung tumors. Oncogene. 2006;25(14):2105–12. [PubMed]
106. Simon DM, et al. Epithelial cell PPARgamma is an endogenous regulator of normal lung maturation and maintenance. Proc Am Thorac Soc. 2006;3(6):510–1. [PubMed]
107. Reynolds SD, et al. Conditional stabilization of beta-catenin expands the pool of lung stem cells. Stem Cells. 2008;26(5):1337–46. [PMC free article] [PubMed]
108. Yang LT, Li WY, Kaartinen V. Tissue-specific expression of Cre recombinase from the Tgfb3 locus. Genesis. 2008;46(2):112–8. [PMC free article] [PubMed]
109. Tsao PN, et al. Notch signaling prevents mucous metaplasia in mouse conducting airways during postnatal development. Development. 2011;138(16):3533–43. [PubMed]
110. Giangreco A, et al. Stem cells are dispensable for lung homeostasis but restore airways after injury. Proc Natl Acad Sci U S A. 2009;106(23):9286–91. [PubMed]
111. Hong KU, et al. Clara cell secretory protein-expressing cells of the airway neuroepithelial body microenvironment include a label-retaining subset and are critical for epithelial renewal after progenitor cell depletion. Am J Respir Cell Mol Biol. 2001;24(6):671–81. [PubMed]
112. Blatt EN, et al. Forkhead transcription factor HFH-4 expression is temporally related to ciliogenesis. Am J Respir Cell Mol Biol. 1999;21(2):168–76. [PubMed]
113. Brody BL, et al. Age-related macular degeneration: a randomized clinical trial of a self-management intervention. Ann Behav Med. 1999;21(4):322–9. [PubMed]
114. Cruz C, et al. Foxj1 regulates floor plate cilia architecture and modifies the response of cells to sonic hedgehog signalling. Development. 2010;137(24):4271–82. [PubMed]
115. Yu X, et al. Foxj1 transcription factors are master regulators of the motile ciliogenic program. Nat Genet. 2008;40(12):1445–53. [PubMed]
116. Ostrowski LE, et al. Targeting expression of a transgene to the airway surface epithelium using a ciliated cell-specific promoter. Mol Ther. 2003;8(4):637–45. [PubMed]
117. Zhang Y, et al. A transgenic FOXJ1-Cre system for gene inactivation in ciliated epithelial cells. Am J Respir Cell Mol Biol. 2007;36(5):515–9. [PMC free article] [PubMed]
118. Rawlins EL, Hogan BL. Ciliated epithelial cell lifespan in the mouse trachea and lung. Am J Physiol Lung Cell Mol Physiol. 2008;295(1):L231–4. [PubMed]
119. Turner J, et al. Goblet cells are derived from a FOXJ1-expressing progenitor in a human airway epithelium. Am J Respir Cell Mol Biol. 2011;44(3):276–84. [PubMed]
120. Rock JR, Randell SH, Hogan BL. Airway basal stem cells: a perspective on their roles in epithelial homeostasis and remodeling. Dis Model Mech. 2010;3(9-10):545–56. [PubMed]
121. Cole BB, et al. Tracheal Basal cells: a facultative progenitor cell pool. Am J Pathol. 2010;177(1):362–76. [PubMed]
122. Byrne C, Fuchs E. Probing keratinocyte and differentiation specificity of the human K5 promoter in vitro and in transgenic mice. Mol Cell Biol. 1993;13(6):3176–90. [PMC free article] [PubMed]
123. Bruen KJ, et al. Real-time monitoring of keratin 5 expression during burn re-epithelialization. J Surg Res. 2004;120(1):12–20. [PubMed]
124. Rock JR, Hogan BL. Epithelial Progenitor Cells in Lung Development, Maintenance, Repair, and Disease. Annu Rev Cell Dev Biol. 2011 [PubMed]
125. Ghosh M, et al. Context-Dependent Differentiation of Multipotential Keratin 14-Expressing Tracheal Basal Cells. Am J Respir Cell Mol Biol. 2010 [PMC free article] [PubMed]
126. Vasioukhin V, et al. The magical touch: genome targeting in epidermal stem cells induced by tamoxifen application to mouse skin. Proc Natl Acad Sci U S A. 1999;96(15):8551–6. [PubMed]
127. Hong KU, et al. Basal cells are a multipotent progenitor capable of renewing the bronchial epithelium. Am J Pathol. 2004;164(2):577–88. [PubMed]
128. Xie W, et al. Conditional expression of the ErbB2 oncogene elicits reversible hyperplasia in stratified epithelia and up-regulation of TGFalpha expression in transgenic mice. Oncogene. 1999;18(24):3593–607. [PubMed]
129. Ito T, et al. Basic helix-loop-helix transcription factors regulate the neuroendocrine differentiation of fetal mouse pulmonary epithelium. Development. 2000;127(18):3913–21. [PubMed]
130. Linnoila RI, et al. Loss of GFI1 impairs pulmonary neuroendorine cell proliferation, but the neuroendocrine phenotype has limited impact on post-naphthalene airway repair. Lab Invest. 2007;87(4):336–44. [PMC free article] [PubMed]
131. Wikenheiser-Brokamp KA. Rb family proteins differentially regulate distinct cell lineages during epithelial development. Development. 2004;131(17):4299–310. [PubMed]
132. Neptune ER, et al. Targeted disruption of NeuroD, a proneural basic helix-loop-helix factor, impairs distal lung formation and neuroendocrine morphology in the neonatal lung. J Biol Chem. 2008;283(30):21160–9. [PMC free article] [PubMed]
133. Johnston D, Hatzis D, Sunday ME. Expression of v-Ha-ras driven by the calcitonin/calcitonin gene-related peptide promoter: a novel transgenic murine model for medullary thyroid carcinoma. Oncogene. 1998;16(2):167–77. [PubMed]
134. Sutherland KD, et al. Cell of origin of small cell lung cancer: inactivation of Trp53 and Rb1 in distinct cell types of adult mouse lung. Cancer Cell. 2011;19(6):754–64. [PubMed]
135. Reynolds SD, et al. Neuroepithelial bodies of pulmonary airways serve as a reservoir of progenitor cells capable of epithelial regeneration. Am J Pathol. 2000;156(1):269–78. [PubMed]
136. Singh SR, et al. Can lineage-specific markers be identified to characterize mesenchyme-derived cell populations in the human airways? Am J Physiol Lung Cell Mol Physiol. 2010;299(2):L169–83. [PubMed]
137. Quaggin SE, et al. The basic-helix-loop-helix protein pod1 is critically important for kidney and lung organogenesis. Development. 1999;126(24):5771–83. [PubMed]
138. Chapman DL, et al. Expression of the T-box family genes, Tbx1-Tbx5, during early mouse development. Dev Dyn. 1996;206(4):379–90. [PubMed]
139. del Moral PM, et al. Differential role of FGF9 on epithelium and mesenchyme in mouse embryonic lung. Dev Biol. 2006;293(1):77–89. [PubMed]
140. Sgantzis N, et al. HuR controls lung branching morphogenesis and mesenchymal FGF networks. Dev Biol. 2011;354(2):267–79. [PubMed]
141. Yu K, et al. Conditional inactivation of FGF receptor 2 reveals an essential role for FGF signaling in the regulation of osteoblast function and bone growth. Development. 2003;130(13):3063–74. [PubMed]
142. Yin Y, et al. An FGF-WNT gene regulatory network controls lung mesenchyme development. Dev Biol. 2008;319(2):426–36. [PMC free article] [PubMed]
143. Abler LL, Mansour SL, Sun X. Conditional gene inactivation reveals roles for Fgf10 and Fgfr2 in establishing a normal pattern of epithelial branching in the mouse lung. Dev Dyn. 2009;238(8):1999–2013. [PMC free article] [PubMed]
144. De Langhe SP, et al. Formation and differentiation of multiple mesenchymal lineages during lung development is regulated by beta-catenin signaling. PLoS ONE. 2008;3(1):e1516. [PMC free article] [PubMed]
145. Saga Y, et al. MesP1 is expressed in the heart precursor cells and required for the formation of a single heart tube. Development. 1999;126(15):3437–47. [PubMed]
146. Strutz F, et al. Identification and characterization of a fibroblast marker: FSP1. J Cell Biol. 1995;130(2):393–405. [PMC free article] [PubMed]
147. Iwano M, et al. Evidence that fibroblasts derive from epithelium during tissue fibrosis. J Clin Invest. 2002;110(3):341–50. [PMC free article] [PubMed]
148. Trimboli AJ, et al. Direct evidence for epithelial-mesenchymal transitions in breast cancer. Cancer Res. 2008;68(3):937–45. [PubMed]
149. Imai T, et al. Impaired adipogenesis and lipolysis in the mouse upon selective ablation of the retinoid X receptor alpha mediated by a tamoxifen-inducible chimeric Cre recombinase (Cre-ERT2) in adipocytes. Proc Natl Acad Sci U S A. 2001;98(1):224–8. [PubMed]
150. West J, et al. Pulmonary hypertension in transgenic mice expressing a dominant-negative BMPRII gene in smooth muscle. Circ Res. 2004;94(8):1109–14. [PubMed]
151. Kuhbandner S, et al. Temporally controlled somatic mutagenesis in smooth muscle. Genesis. 2000;28(1):15–22. [PubMed]
152. Owens GK, Kumar MS, Wamhoff BR. Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev. 2004;84(3):767–801. [PubMed]
153. Woodcock-Mitchell J, et al. Alpha-smooth muscle actin is transiently expressed in embryonic rat cardiac and skeletal muscles. Differentiation. 1988;39(3):161–6. [PubMed]
154. Mitchell JJ, et al. Smooth muscle cell markers in developing rat lung. Am J Respir Cell Mol Biol. 1990;3(6):515–23. [PubMed]
155. Perl AK, Gale E. FGF signaling is required for myofibroblast differentiation during alveolar regeneration. Am J Physiol Lung Cell Mol Physiol. 2009;297(2):L299–308. [PubMed]
156. Mitchell J, et al. Alpha-smooth muscle actin in parenchymal cells of bleomycin-injured rat lung. Lab Invest. 1989;60(5):643–50. [PubMed]
157. Thet LA, Parra SC, Shelburne JD. Sequential changes in lung morphology during the repair of acute oxygen-induced lung injury in adult rats. Exp Lung Res. 1986;11(3):209–28. [PubMed]
158. Wu Z, et al. Detection of epithelial to mesenchymal transition in airways of a bleomycin induced pulmonary fibrosis model derived from an alpha-smooth muscle actin-Cre transgenic mouse. Respir Res. 2007;8:1. [PMC free article] [PubMed]
159. Wendling O, et al. Efficient temporally-controlled targeted mutagenesis in smooth muscle cells of the adult mouse. Genesis. 2009;47(1):14–8. [PubMed]
160. Regan CP, Manabe I, Owens GK. Development of a smooth muscle-targeted cre recombinase mouse reveals novel insights regarding smooth muscle myosin heavy chain promoter regulation. Circ Res. 2000;87(5):363–9. [PubMed]
161. Xin HB, et al. Smooth muscle expression of Cre recombinase and eGFP in transgenic mice. Physiol Genomics. 2002;10(3):211–5. [PubMed]
162. Wilm B, et al. The serosal mesothelium is a major source of smooth muscle cells of the gut vasculature. Development. 2005;132(23):5317–28. [PubMed]
163. Que J, et al. Mesothelium contributes to vascular smooth muscle and mesenchyme during lung development. Proc Natl Acad Sci U S A. 2008;105(43):16626–30. [PubMed]
164. Morimoto M, et al. Canonical Notch signaling in the developing lung is required for determination of arterial smooth muscle cells and selection of Clara versus ciliated cell fate. J Cell Sci. 2010;123(Pt 2):213–24. [PubMed]
165. Braren R, et al. Endothelial FAK is essential for vascular network stability, cell survival, and lamellipodial formation. J Cell Biol. 2006;172(1):151–62. [PMC free article] [PubMed]
166. Proctor JM, et al. Vascular development of the brain requires beta8 integrin expression in the neuroepithelium. J Neurosci. 2005;25(43):9940–8. [PMC free article] [PubMed]
167. Koni PA, et al. Conditional vascular cell adhesion molecule 1 deletion in mice: impaired lymphocyte migration to bone marrow. J Exp Med. 2001;193(6):741–54. [PMC free article] [PubMed]
168. Kisanuki YY, et al. Tie2-Cre transgenic mice: a new model for endothelial cell-lineage analysis in vivo. Dev Biol. 2001;230(2):230–42. [PubMed]
169. DasGupta R, Fuchs E. Multiple roles for activated LEF/TCF transcription complexes during hair follicle development and differentiation. Development. 1999;126(20):4557–68. [PubMed]
170. Maretto S, et al. Mapping Wnt/beta-catenin signaling during mouse development and in colorectal tumors. Proc Natl Acad Sci U S A. 2003;100(6):3299–304. [PubMed]
171. Lohi M, Tucker AS, Sharpe PT. Expression of Axin2 indicates a role for canonical Wnt signaling in development of the crown and root during pre- and postnatal tooth development. Dev Dyn. 2010;239(1):160–7. [PubMed]
172. Al Alam D, et al. Contrasting Expression of Canonical Wnt Signaling Reporters TOPGAL, BATGAL and Axin2 during Murine Lung Development and Repair. PLoS One. 2011;6(8):e23139. [PMC free article] [PubMed]
173. Ferrer-Vaquer A, et al. A sensitive and bright single-cell resolution live imaging reporter of Wnt/ss-catenin signaling in the mouse. BMC Dev Biol. 2010;10:121. [PMC free article] [PubMed]
174. Duncan AW, et al. Integration of Notch and Wnt signaling in hematopoietic stem cell maintenance. Nat Immunol. 2005;6(3):314–22. [PubMed]
175. Mizutani K, et al. Differential Notch signalling distinguishes neural stem cells from intermediate progenitors. Nature. 2007;449(7160):351–5. [PubMed]
176. Rock JR, et al. Notch-dependent differentiation of adult airway Basal stem cells. Cell Stem Cell. 2011;8(6):639–48. [PMC free article] [PubMed]
177. Vooijs M, et al. Mapping the consequence of Notch1 proteolysis in vivo with NIP-CRE. Development. 2007;134(3):535–44. [PMC free article] [PubMed]
178. Jackson EL, et al. Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras. Genes Dev. 2001;15(24):3243–8. [PubMed]
179. Gregory LG, et al. Overexpression of Smad2 drives house dust mite-mediated airway remodeling and airway hyperresponsiveness via activin and IL-25. Am J Respir Crit Care Med. 2010;182(2):143–54. [PMC free article] [PubMed]
180. Wunderlich S, et al. Type II pneumocyte-restricted green fluorescent protein expression after lentiviral transduction of lung epithelial cells. Hum Gene Ther. 2008;19(1):39–52. [PubMed]
181. Hendrickson B, et al. Development of lentiviral vectors with regulated respiratory epithelial expression in vivo. Am J Respir Cell Mol Biol. 2007;37(4):414–23. [PMC free article] [PubMed]
182. Liu X, et al. Analysis of adeno-associated virus progenitor cell transduction in mouse lung. Mol Ther. 2009;17(2):285–93. [PMC free article] [PubMed]
183. Lo B, et al. Alveolar epithelial type II cells induce T cell tolerance to specific antigen. J Immunol. 2008;180(2):881–8. [PubMed]
184. Li H, et al. Cre-mediated recombination in mouse Clara cells. Genesis. 2008;46(6):300–7. [PubMed]
185. Malkoski SP, et al. Keratin promoter based gene manipulation in the murine conducting airway. Int J Biol Sci. 2010;6(1):68–79. [PMC free article] [PubMed]
186. Long MA, Rossi FM. Silencing inhibits Cre-mediated recombination of the Z/AP and Z/EG reporters in adult cells. PLoS One. 2009;4(5):e5435. [PMC free article] [PubMed]
187. Beard C, et al. Efficient method to generate single-copy transgenic mice by site-specific integration in embryonic stem cells. Genesis. 2006;44(1):23–8. [PubMed]
188. Sisson TH, et al. Targeted injury of type II alveolar epithelial cells induces pulmonary fibrosis. Am J Respir Crit Care Med. 2010;181(3):254–63. [PMC free article] [PubMed]
189. Stanger BZ, Tanaka AJ, Melton DA. Organ size is limited by the number of embryonic progenitor cells in the pancreas but not the liver. Nature. 2007;445(7130):886–91. [PubMed]