Figure 1.
Schematic view of the Ifitm1 targeting strategy by homologous recombination in mouse ES cells.
(A) Shows the scheme of the linearized targeting vector used for electroporation in mouse ES cells. The coding sequence of the lacZ reporter gene (lacZ) was inserted in frame into the first codon of the Ifitm1 gene. The three Ifitm1 exons are indicated as I, II, and III. The lacZ gene is followed by a poly-adenylation signal (bGHpA) and a neomycin/kanamycin selection cassette (neo/kan) that was flanked by loxP sites (L) for subsequent deletion of the cassette. The backbone of the vector (vb) included the coding sequence for the diphtheria toxin fragment A (DTA) to support the selection of clones carrying the homologous recombination of the targeting vector. The location of the probe used for Southern blotting (sp) and PvuII restriction sites (P) are also indicated. The PvuII restriction sites that are relevant for the diagnostic restriction fragments in Southern blots in combination with DNA probe sp are marked with an underline (P). (B) Shows a scheme of the wildtype Ifitm1 locus on mouse chromosome 7. The three Ifitm1 exons (I, II, and III) are indicated as boxes. Black shading in the boxes indicates the coding sequence of Ifitm1. The genomic region that was amplified by PCR to genotype the Ifitm1wt allele is indicated (wt-PCR). (C) Shows the Ifitm1 locus following homologous recombination in mouse ES cells. As result of recombination, the complete Ifitm1 coding sequence is replaced by the lacZ reporter gene and the selection cassette (lacZ-bGHpA-Lneo/kanL). The genomic region that is covered by the homologous sequence of the targeting vector (tv) is designated. The genomic region that was amplified by PCR to genotype the targeted Ifitm locus in the electroporated ES cells following G418 selection is indicated (ES-PCR). (D) Shows a scheme of the Ifitm1tm1IEG loss-of-function allele that was generated by expression of the Cre recombinase and subsequent deletion of the neo/kan selection cassette from the targeted Ifitm1 allele shown in (C). As result of this excision, a single loxP site (L) is left behind as indicated. The genomic region that was amplified by PCR to genotype the targeted Ifitmtm1IEG loss-of-function allele in mice obtained from matings between chimeric mice and Cre expressing mice is indicated (flox-ko-PCR). The size bar (bottom left) indicates 1 kb length.
Table 1.
Most significantly over-represented Gene Ontology (GO) terms among regulated genes in the comparative transcriptome analysis of Ifitm1tm1IEG/tm1IEG versus Ifitm1wt/wt lungs and list of regulated genes annotated with the respective GO terms.
Figure 2.
LacZ expression pattern in Ifitm1tm1IEG/wt embryos from E8.5 to E11.5.
(A – E) At E8.5 strong expression was found in the future brain and the neural tube. Weak expression was detected in the allantois. (F – I) At E9.5 lacZ expression was detected in the future brain, the primordial gut, the somites and the presomitic mesoderm. The anlage of the lung was the anterior boundary of reporter gene expression in the trunk region (see dashed line). (J – M) At E10.5 the reporter gene expression pattern was similar to the one at E9.5. The expression in the ventral brain was extended along the anterior-posterior axis. (N – Q) At E11.5 lacZ expression decreased in the PSM and in somites. Weak expression was detected in the brain and a new expression domain developed in the urogenital ridge. Abbreviations: a – allantois, b – brain, g – primordial gut, la – anlage of the lung, nt – neural tube, PSM – presomitic mesoderm, s – somite, ur – urogenital ridge.
Figure 3.
LacZ expression pattern in E12.5 to E14.5 Ifitm1tm1IEG/wt mouse embryos.
A–D: E12.5: lacZ was highly expressed in the floor plate of the neural tube and the urogenital ridge. E – H: E13.5: several new embryonic lacZ expression domains appeared in the brain, the intestine, the mucosa of the oropharynx, the olfactory epithelium, the papilla of the tongue, the sympathetic ganglion and the thymus. Background staining due to unspecific X-Gal staining was found in brain. I – L: E14.5: additional expression domains were detected in the primordia of hair follicles as well as in the ovary. Background staining in wildtype littermates was found in the brain, the inner ear, and the paramesonephric duct. b – brain, fp – floor plate, h – primordium of hair follicle, i – intestine, ie – inner ear, o – oropharynx, oe – olfactory epithelium, ov – ovary, p – papilla of the tongue, pd - paramesonephric duct, sg – sympathetic ganglion, th – thymus, ur – urogenital ridge.
Figure 4.
Histological sections of LacZ stained Ifitm1tm1IEG/wt mouse embryo at E12.5.
Lines in the bleached whole embryo (left panel) indicate planes of histological sections in panels A and B on the right. (A) LacZ expression was found in cells of the floor plate (fp) of the neural tube. (B) Gene expressions in cells of the genital ridge (gr) is weak and strong in the tubuli of the metanephros (tu). Scale bars indicate 100 µm.
Figure 5.
Histological sections of LacZ stained Ifitm1tm1IEG/wt mouse embryo at E13.5.
Lines in the bleached whole embryo (panel at the top left) indicate planes of histological sections (A to E). (A) LacZ expression was found in the epithelium of the cerebral aqueduct (eca), (B and B’) the sympathetic ganglion (sg), (C) in the papillae of the tongue (p) and the epithelium of the oropharynx (o), (D) the olfactory epithelium (oe), (E) the thymus (th), (F) primordia of the teeth (pt), (G) cells of the floor plate (fp) of the neural tube, (H) the genital ridge (gr) and the (I) metanephros (m). Scale bars indicate 100 µm.
Figure 6.
Histological sections of LacZ stained mouse Ifitm1tm1IEG/wt embryo at E14.5. Lines
in the bleached whole embryo (panel at the op left) indicate planes of histological sections (A to M). (A) LacZ expression was found in sympathetic ganglia (sg), (B) the eyelids (e), (C) the epithelium of the oropharynx (o), (D) the olfactory epithelium (oe), (E) primordia of the teeth (pt), (F) the thymus (th), (G) the serous glands associated with the nasal septum, (H) primordia of hair follicles (h), (I) in cells of the floor plate (fp) of the neural tube, (J) mesenchymal cells at the inner side of the limbs (l), (K and K’) cells of the outer wall of the intestine (i), (L) cells of the ovary (ov), and (M) in cells of the hindgut (hg). Scale bars indicate 100 µm.
Figure 7.
Ifitm1/IFITM1 expression in mouse and human lung.
(A and B) Detection of Ifitm1 expression in adult mouse lung by whole organ X-Gal staining. LacZ expression was detected in the bronchia of the lung of Ifitm1tm1IEG/wt adult mice (A) but not in wildtype lungs (B). (C and D) Subsequent Histological sections of stained Ifitm1tm1IEG/wt mouse lungs revealed lacZ expression in the cells of the bronchial epithelium of Ifitm1tm1IEG/wt mice. (E) By immunohistochemistry on human adult lung tissue, IFITM1 expression was detected in the columnar- and in the basal cells of the bronchial epithelium. (F) Immunohistochemical detection of CYTOKERATIN-7 (CK7) was used to identify columnar cells of the bronchial epithelium. Scale bars indicate 100 µm.
Figure 8.
Reporter gene expression in thymus from adult Ifitm1tm1IEG/wt and wildtype mice.
(A and B) Whole organ X-Gal staining of the thymus of heterozygous knockout animals exhibited lacZ expression whereas thymus of wildtype littermate mice did not stain for X-Gal. (C and D) Histological sections of stained thymus revealed cells of the medulla expressing the lacZ reporter gene in Ifitm1tm1IEG/wt mice (C) but not in thymus from wildtype littermate mice (D). Scale bars indicate 100 µm.
Figure 9.
IFITM1 immunohistochemistry on human lung tissue (40x).
(A and D) IFITM1 is expressed in non-neoplastic alveolar and bronchial epithelia. (B and E) The IFITM1 gene is highly overexpressed in squamous cell carcinomas of the human lung and (C and F) in adenocarcinomas of the human lung.