Conditional inactivation of Emilin1 and Col6a1 genes

Conditional gene expression methods are important approaches for examining the function of particular genes in development and disease. In particular, a lentiviral vector system for RNAi- mediated in vivo silencing of Col6a1 and a Cre-loxP based procedure for conditional-inducible knockout (KO) of Emilin1 were used in this work. The use of lentiviral vectors can accelerate the generation of animals with substantial suppression of gene expression in an inducible way. Some authors have observed limitations of the technique due to low efficiency of transgenesis and mosaicism in transgenic mice. We have generated several transgenic mouse lines expressing siRNAs that target the α1(VI) mRNA. Characterization of the different lines and comparison of the phenotypes with that of Col6a1 knockout mice have allowed a systematic evaluation of the different factors affecting silencing of Col6a1, a gene of the extracellular matrix with a complex pattern of tissue-specific expression. The results, obtained with vectors pLVTHM and pLVPT-rtTRKRAB, point out three parameters as major determinants of the efficiency of interference: the choice of interfering sequence, the number of proviral copies integrated into the mouse genome and the site of integration of the provirus. A lentiviral vector (pLVPT-rtTRKRAB) with doxycycline inducible production of shRNA was also tested. Control of expression by the drug was stringent in many tissues; however, in some tissues turning off of shRNA synthesis was not complete. The data support the application of the lentiviral vectors used here in transgenesis (Frka, Facchinello, et al., 2009). Emilin1 is a gene coding for a protein, Emilin-1, of the elastic extracellular matrix expressed in interstitial connective tissue and in the cardiovascular system starting form early stages of embryonic development to adulthood. Emilin1 null mice display reduced diameter of blood vessels and arterial hypertension. The protein regulates the bioavailability of TGF-β, a cytokine with major effects on the cardiovascular system. Specifically, it has been shown that Emilin1 inhibits proteolysis of the proTGF-β precursor to LAP/TGF-β, a complex from which the growth factor can be subsequently released for receptor binding. In the absence of Emilin1, the amount of active TGF-β is increased, reducing the proliferation rate of smooth muscle cells and the diameter of blood vessels. To establish whether the Emilin1-/- phenotype is the result of a developmental defect or the function of the protein is required for the regulation of blood pressure and arterial structure also in the adult, a conditional gene targeting procedure was used to inactivate Emilin1 in a tissue and time-specific manner. The genetic set up of the transgenic mouse model included the use of floxed Emilin1, CreERT2 (a tamoxifen inducible Cre recombinase) tissue-specific drivers and Rosa26R-lacZ, an inducible reporter for histological visualization of gene rearrangement. A targeting vector was synthesized in which exons 1-2 of the Emilin1 gene were flanked by loxP sites (floxed allele). The correct integration of the targeting construct in embryonic stem (ES) cell clones was confirmed by Southern blot and PCR analyses. Four of the ES cell clones were subsequently injected into blastocysts resulting in chimeric mice with 10-100% chimerism. The CreERT2 gene was driven by the Emilin1 or the smooth muscle myosin heavy chain (SMMHC) promoters in order to be expressed in cells active in Emilin1 synthesis or in smooth muscle cells respectively. After tamoxifen administration, activity of both promoters was evident in vascular and visceral smooth muscle cells, where SMMHC-CreERT2 induced recombination more strongly than Emilin1-CreERT2. PCR and RT-PCR analyses confirmed that the SMMHC-CreERT2 was expressed and efficiently excised the loxP flanked sequences in the Emilin1flox/wt locus sufficiently to reduce Emilin1 mRNA expression. Emilin1flox/flox mice appeared and bred normally and showed no difference in Emilin1 mRNA expression and in blood pressure levels as compared to controls, confirming that introduction of the loxP sites did not interfere with regulation of the gene. Moreover preliminary data showed that adult animals of the double transgenic lines Emilin1flox/flox and SMMHC-Cre-ERT2 treated with tamoxifen displayed increased blood pressure. This result suggests that hypertension in Emilin1flox/flox mice is not due to a developmental defect of blood vessels, but to the lack of a continuous effect of Emilin-1 on blood pressure regulation even in the adult.