Mice bearing a homozygous floxed 3 laminin subunit allele in the absence of the Neo cassette were further bred to remove the transgene. significant increase in the collagen content of the lungs. We conclude that the loss of 3 laminin in the alveolar epithelium results in an increase in lung collagen, which confers resistance to mechanical injury. floxed mice We generated a vector targeting exon 42 of the Bestatin Methyl Ester mouse gene (the murine equivalent of exon 41 of the human gene) by positioning sequences Rabbit Polyclonal to SLC6A15 3 kb upstream and 4 kb downstream of exon 42 (Fig. 1A). Deletion of this exon will result in loss of the two main splice isoforms encoded by the gene (the 3a and 3b laminin subunits) (Ryan et al., 1999). This vector was launched into embryonic stem cells (ESCs) through electroporation, and the cells were selected and screened for homologous recombination by southern blotting. These cells were injected into blastocysts, which were then implanted into mice. The chimeric offspring were mated to C57BL/6 mice and ESC-derived progeny were sequentially bred to produce mice homozygous for the floxed 3 laminin subunit allele. To avoid the possibility that the Neo cassette, which contains strong regulatory regions, might influence expression of the targeted gene or it neighbors, we removed it by mating the homozygous floxed mice to mice. Mice bearing a homozygous floxed 3 laminin subunit allele in the Bestatin Methyl Ester absence of the Neo cassette were further bred to remove the transgene. DNA isolated from your tails of five littermates resulting from crossing animals exhibiting germline transmission of the floxed allele was digested with sites (Fig. 1B). The expected size for the wild-type (wt) fragment is usually 26 kb and for the flox fragment is usually 9.8 kb. Genomic DNA was isolated from your lungs of wild-type mice infected with null computer virus, and mice infected with null computer virus or Cre-encoding computer virus. PCR primers were designed to amplify within intron 40 through intron 42, a region flanking the designed sites. We amplified the expected 950-bp product from your wild-type lung genomic DNA and an 1100-bp product from your lung genomic Bestatin Methyl Ester DNA from mice treated with control computer virus, indicating the insertion of the sites. Using the same primers, we amplified an additional 500-bp product from lung genomic DNA from your mice treated with Cre computer virus, indicating excision of the targeted sequence (Fig. 1C). To ensure that the knockdown did not result in the production of an N-terminal fragment, we designed primer sequences to amplify short regions specifically within the and regions and within a region common to both, downstream of the deleted exon (supplementary material Fig. S1). In RNA obtained from alveolar type II cells isolated from mice treated with Ad-Null or Ad-Cre 60 days earlier, all three products were significantly reduced confirming a knockdown of and and suggesting that no N-terminal laminin fragment is usually produced (Fig. 1D). Open in a separate windows Fig. 1. Generation of the mouse. (A) Diagram of part of the vector that targets exon 42 of the mouse gene. (B) DNA isolated from tails of five littermates resulting from crossing animals exhibiting germline transmission of the floxed allele was digested with sites. The expected size for the wild-type (wt) fragment is usually 26 kb and for the flox fragment is usually 9.8 kb. (C) Genomic DNA was isolated from your lungs of null virus-infected wild-type mice (wt), null-virus-infected mice (fl/fl Null) and Cre computer virus infected mice (fl/fl Cre) and subjected to PCR using primers flanking the designed region. (D) mice were treated intratracheally with a null adenovirus (Null) or an adenovirus encoding Cre recombinase (Cre) and 60 days later alveolar type II cells were isolated from your mice from which RNA was isolated for measurement (qRT-PCR) of short regions of the gene specific to (3a) or (3b) transcripts or those common to and (3a/b) (also observe supplementary material Fig. S1). Lung-specific knockdown of 3 laminin mice were treated with Ad-Cre or Ad-Null and 30 days, 60 days or 6 months later the lungs were harvested, homogenized and the abundance of the 3 laminin subunit was detected by immunoblotting. Using -galactosidase Cre reporter mice, we have previously shown that this dose of adenovirus.