A general method for gene knockdown in mice by using lentiviral vectors expressing small interfering RNA

Abstract

We describe the use of lentiviral vectors expressing small interfering RNAs (siRNAs) to knock down the expression of specific genes in vitro and in vivo. A lentiviral vector capable of generating siRNA specific for GFP after transduction of 293T-GFP cell lines showed no GFP fluorescence. Furthermore, no GFP-specific RNA could be detected. When eggs from GFP-positive transgenic mice were transduced with lentivirus-expressing siGFP virus, reduced fluorescence could be seen in blastocysts. More interestingly, pups from F(1) progeny, which expressed siGFP, showed considerably diminished fluorescence and decreased GFP. We propose that an approach of combining transgenesis by lentiviral vectors expressing siRNAs can be used successfully to generate a large number of mice in which the expression of a specific gene(s) can be down-regulated substantially. We believe that this approach of generating "knockdown" mice will aid in functional genomics.

Figure 1

Transfection by the siGFP plasmid. (a) Sequence and putative folding of siGFP oligonucleotide used to target the GFP gene. The 20-nt sequence of the target transcript separated by a short spacer (shown as a loop) from reverse complement of the same sequence is shown. The termination sequence consists of five thymidines (T5). (b) Transfection of 293T cells with 0.2 μg of GFP-expressing plasmid together with 1 μg of a plasmid expressing siGFP or siHp53. A lack of GFP expression only in the presence of siGFP-expressing plasmid can be seen. Photographs were taken with a Zeiss microscope (×5). (c) Twenty micrograms of RNA prepared from transfected cells were electrophoresed on a denaturing gel and hybridized to radiolabeled GFP, human p53, and β-actin probes. Specific silencing of GFP and human p53 RNA was noticed only when the relevant specific siRNA was used.

Figure 2

LV-siGFP virus transduction. (a) Schematic drawing of lentiviral vector containing an siGFP cassette capable of generating siGFP-RNA. Shown also is the construct capable of generating two copies of siRNA after reverse transcription and integration. (b) Fifty thousand 293T cells were transduced with increasing amounts of LV-siGFP (15–375 ng of p24 per well). Fluorescence-activated cell-sorter analysis was performed to detect fluorescence. (c) Complete extinction of GFP expression by transduction of 80 ng (p24) of LV-siGFP virus in 5 × 10⁴ 293T-GFP-positive cells (×30). (d) 293T-GFP cells were transduced with increasing amounts of LV-siGFP (15–375 ng of p24 per well). Seventy-two hours later, whole-cell extracts were prepared, separated on 10% SDS/PAGE, and immunoblotted with polyclonal antibodies to GFP. As a control, 293T-GFP cells were also transduced with increasing amounts of LV-siHp53 and probed with GFP antibodies. β-Actin was used as a loading control.

Figure 3

Generation of GFP knockdown mice. (a) Decreased fluorescence of GFP in blastocytes transduced with LV-siGFP (♂, filled green depicts GFP transgenic male mouse; ♀, wild-type female). (b) F₀ animals were analyzed by PCR of genomic DNA using U3 primers. The U3 primers amplified the H1-siGFP cassette cloned in the U3 region in the LTR. The siGFP-positive F₀ mice (F₀-2 + F₀-4) are indicated in red. (c) PCR of genomic DNA from F₁ pups using U3-H1 primers. The combination of U3 forward and H1 reverse amplifies the H1 promoter portion of the H1-siGFP cassette. The positive F₁-36 pup is shown in red. Persistence of the GFP allele was confirmed by PCR using GFP primers. (d) Shining with fluorescence lamp of F₁ littermate pups at day 3 of age (pictures were taken with a digital camera). (e) Embryonic day-13 embryos were harvested from pregnant female F₀-4, and GFP sequences were detected using GFP-specific PCR primers. F₁ embryos positive for integrated copies of the siGFP cassette using U3-H1 primers are shown. The F₁-6 embryo positive for both GFP and siGFP is shown in red. (f) Protein extracts from embryonic day-13 embryos were analyzed by probing with a GFP antibody. Shown are embryos 1, 6, and 7. Reprobing with β-actin antibody was used as a loading control.