Expression Analysis of CB2-GFP BAC Transgenic Mice
Fig 1
Construction of CB2-GFP BAC transgene and expression in various transgenic mouse tissues.
(a) Schematic diagram of GFP-FRT-Neo® BAC modification fragment and homologous recombination into the wild-type Cnr2 BAC depicts Cnr2 exons (black rectangles), ORF (open box), and GFP-FRT-Neo® fusion reporter (green/blue). Cnr2 homology arms (each ∼50 bp) are shown on either side of the insert sequence. Introduction of the GFP-FRT-Neo® cassette leads to a replacement of the Cnr2 ORF without affecting any putative Cnr2 promoter sequences. Excision of the Neo® cassette after integration to derive the final CB2-GFPTg BAC transgene is shown. Restriction enzymes and a genomic probe (black bar) used for Southern blot analysis are indicated. Exons are represented as rectangles and FRT sites as triangles. P: PstI; A: AseI; C: ClaI. (b) Screening for transgene integration by PCR. Integration of the CB2-GFPTg BAC into the genomic mouse DNA was confirmed by PCR with specific primers (eGFP_Aat_F2 and eGFP_R2) as indicated with arrows in a. Three independent founders were identified (asterisks) by the detection of an additional band representing the GFP. (c) Comparison of GFP and CB2 mRNA expression in brain, spleen and thymus relative to HPRT reference gene expression. In brain tissue samples neither GFP nor CB2 mRNA expression was detectable. Thymus showed moderate expression of both, GFP and CB2, whereas spleen tissue revealed highest expression of GFP and CB2. n = 4. (d) Representative western blot analysis of GFP protein expression in brain, thymus and spleen in CB2-GFPTg and WT mice. CB2-GFPtg mice showed moderate GFP protein expression in thymus and high GFP protein expression in spleen. No detectable GFP protein expression in brain tissue samples as well as in samples of WT littermates. GAPDH was used as loading control.