In vitro and in vivo studies have repeatedly demonstrated the limitations and potential toxicity of various genes (proteins) used for both labeling cells (eg, with green fluorescent protein [GFP], b-galactosidase, and luciferase) or for the deletion/addition of mutation of genes (eg, reverse tetracycline transactivator protein [rtTA], tetracycline transactivator protein [tTA], Cre-recombinase [Cre], or CreER). High levels of the introduced protein can cause endoplasmic reticulum stress, genetic instability, immunologic recognition, and/or disrupt cellular homeostasis. Resultant cell injury, death, or other off-target effects on gene expression may be caused by expression of the transgene. A number of systems for gene addition and deletion in the respiratory epithelium have been developed and widely used for the study of gene function, lung morphogenesis, and function. The Scgb1a1 (Clara Cell Secretory Protein or CCSP) and SFTPC (Surfactant Protein C or SP-C) promoters have been used by our laboratory and others (1, 2). These promoters are highly cell specific and generate robust levels of gene expression. To conditionally express genes in the lung, transgenic mice were produced expressing the reverse tetracycline transactivator that is active when doxycycline is provided to the mouse (3, 4). To target proximal airways, the 2.3-kb rat Scgb1a1 promoter was used to drive the reverse tetracycline activator (CCSP-rtTA transgenic mice). To target distal lung structures, we used the 3.7-kb humanSFTPCpromoter (SP-C-rtTA transgenic mice). Airspace enlargement unrelated to the effects of the transgene were observed in various mouse strains bearing the CCSP-rtTA transgene (line 1)(5–7). The potential for toxicity related to the reverse tetracycline activator, doxycycline, and Cre-recombinase was reviewed previously (7). Such overt rtTA toxicity was not seen in many experiments with the initial 3.7-kb human SP-C-rtTA transgenic mice that have been used in numerous studies. Subsequently, both CCSP-rtTA and SP-C-rtTA mice were bred to a line of (otet) 7CMV-Cre mice that enables doxycyclineregulated expression of Cre-recombinase in the respiratory epithelium in vivo (6, 8). When mated to mice bearing floxed alleles, the addition of doxycycline to chow or drinking water (3) causes excision and recombination of DNA sequences located between engineered loxP recognition sites, causing deletion, mutation, or activation of the appropriately engineered floxed gene in the mouse lung (6–8). This system has been useful in lineage analysis, the study of gene function, and lung development. Initial studies failed to reveal significant misexpression of Cre-recombinase or overt histologic or biological toxicity. As our laboratory has bred the SP-C-rtTA,(otet) 7CMV-Cre mice (primarily maintained in FVBN strain) with other strains bearing floxed alleles and other mouse genetic backgrounds, we have observed off-target effects influencing both lung morphogenesis and perinatal survival. In generating mice in which GP130 receptor, mediating the activation of STAT3, was deleted (9), initial F1 mice were bred, in line, with resultant production of two distinct strains that (1) lacked discernable phenotype, unless injured (9), or (2) died of severe lung pathology at birth. Severe morphologic effects were observed when doxycycline was provided to the dam from approximately Embryonic Day (E) 6 to birth. Toxicity was not dependent on the presence of the floxed allele, but on the presence of both SP-C-rtTA and (otet) 7CMV-Cre, indicating that toxicity was likely dependent on the expression of Cre-recombinase or the combined expression of rtTA and Cre-recombinase in this strain …