Using siRNA technique to generate transgenic animals with spatiotemporal and conditional gene knockdown

Abstract

Based on the RNAi technique, we have developed a new approach that generates transgenic animals capable of mimicking human genetic diseases. The new system is a combination of siRNA with Cre-loxP and tetracycline-on. It has the characteristics of being stable, inheritable, and inducible, with the siRNA able to be transcribed tissue specifically. To support the ability of this new method to generate a model for a disease, we created an ABCA1-deficient mouse line that mimics Tangier disease under controlled conditions. Thus, it should now be possible to rapidly establish human genetic diseases as a whole animal model without the use of embryonic stem cell and gene targeting. This system also provides a tool for pathological and pharmacological studies of aspects peculiar to particular human genetic diseases.

Figure 1

Construction and expression of SIRIUS-Cre system. Schematic diagrams of SIRIUS-ABCA1, AlbTet-Cre, and the resulting reSIRIUS-ABCA1 constructions, all vectors contain LTR (long terminal repeat) and ϕ (virus packaged-signal) fragments. HepG2 cells were co-transfected with plasmids containing SIRIUS-ABCA1 or AlbTet-Cre. After doxycycline treatment and the subsequent recombination, reSIRIUS-ABCA1 was generated. The rtTA, reverse tet-controlled transcriptional activator, binds the TRE (tet-responsive element) in the presence of doxycycline and activates transcription of Cre recombinase under AlbTet promoter controlling. Cre recombinase recognizes loxP sites in the SIRIUS-ABCA1, facilitating precise recombination (dotted line) and activates transcription of ABCA1 (ATP-binding cassette subfamily A member 1) siRNA. These neomycin and hygomycin resistance genes were inserted in SIRIUS-ABCA1 and AlbTet-Cre vectors, respectively. Primers used in this study include CreF, CreD, p1581, and p4214 and are indicated.

Figure 2

Generation of reSIRIUS-ABCA1 and knockdown of ABCA1 via recombination in HepG2 cells after doxycycline (2 μg/ml) treatment. a: PCR analysis of recombined SIRIUS-ABCA1 after treatment (for 2 hours at 37°C) with purified Cre recombinase using specific primers (p1581 and p4214 primers, see Materials and Methods). b: Upper panel, fluorescence pictures of HepG2 cells transfected either with pLEGFP-C1 alone or SIRIUS-ABCA1 alone, or co-transfected with pLEGFP-C1 and AlbTet-Cre or SIRIUS-ABCA1 and AlbTet-Cre, respectively. Photograph displays significant down-regulation of GFP expression in SIRIUS-ABCA1/AlbTet-Cre transfected cells. Lower panel, Western blot of Cre recombinase genomic-PCR analyses to demonstrate the recombination process. Cellular lysates (1: pLEGFP-C1 alone, 2: pLEGFP-C1/AlbTet-Cre, 3: SIRIUS-ABCA1 alone, and 4: SIRIUS-ABCA1/AlbTet-Cre) were prepared 72 hours after transfection with different combinations of vectors. Lower left panel, Western blot analysis of Cre recombinase. There was significant expression of the Cre recombinase in pLEGFP-C1/AlbTet-Cre and SIRIUS-ABCA1/AlbTet-Cre transfected HepG2 cells after doxycycline treatment. Cre polyclonal antibody (Novagen) was used in this study. The commercial Cre recombinase was incorporated as a positive control (con). PCNA was used as an internal control. Lower right panel, genomic-PCR analysis showed that the recombination occurred in HepG2 cells when transfected with SIRIUS-ABCA1/AlbTet-Cre after doxycycline treatment. c: Expression of ABCA1 protein was detected by Western blot in SIRIUS-ABCA1/AlbTet-Cre transfected cells. Result indicates that the ABCA1 expression in SIRIUS-ABCA1/AlbTet-Cre co-transfected cells was progressively decreased according to the time (48, 72, and 96 hours) after transfection. The β-actin was used as an internal control.

Figure 3

The phenotype and symptoms of ABCA1 gene knockdown mice. a: The two separate lines of transgenic animals carrying AlbTet-Cre (F0-A) and SIRIUS-ABCA1 (F0-S) plasmid, respectively. The two separate lines of transgenic mice were mated to generate offspring (F1-SA) that carry both transgenes in a single mouse. F1-SA animals were examined under a fluorescent anatomy-microscopy to visualize the expression pattern of green fluorescent, like in F0-S founders. F1-SA mice at 3 weeks old were supplied with or without doxycycline by drinking and injection as described in Materials and Methods. Liver tissues from F1-SA mice were sectioned and visualized at 96 hours after doxycycline treatment. The fluorescent anatomy-microscopy showed that a reduction in EGFP occurred in F1-SA when treated with doxycycline. b: Cre recombinase and ABCA1-siRNA in various tissues of a F1-SA animal. Upper panel, RT-PCR analysis of Cre-recombinase expression in brain, heat, lung, kidney, and liver tissues. Cre-recombinase was only detected in the liver of a F1-SA mouse. β-Actin was used as an internal control. Lower panel, Northern blot analysis of ABCA1 siRNA in brain, heart, lung, kidney and liver tissues of F1-SA mouse. ABCA1 siRNA was only detected in the liver of two doxycycline-treated F1-SA mice. The different fragments of ABCA1 siRNA are indicated by arrowheads. c: Genomic-PCR analysis of the recombination of SIRIUS-ABCA1 in the livers of doxycycline-treated (treated, T) and -untreated mice (control, C). Left panel, doxycycline-treated F1-SA mice showed the presence of a less than 0.5-kb PCR product same as was shown in the HepG2 cells in Figure 2a. Right panel, Northern blot analysis of ABCA1 in doxycycline-treated and control F1-SA mice. β-actin was used as an internal control. d: Microscopic slides of Oil Red O staining of lipid in the thymus, livers, and kidney derived from non-induced and doxycycline-induced F1-SA mice (Magnification, ×200). Liver specimens exhibited significantly higher levels of cholesteryl ester staining after doxycycline treatment. e: Plasma cholesterol distribution in mice on different days after doxycycline treatment. Fifty μl of plasma were obtained from each of the 12 transgenic animals after doxycycline-treated (5 days to 11 days), together with non-treated (non-) and doxycycline-treated normal control (con-) mice. All three groups were subjected to cholesterol measurement as described in Materials and Methods.