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. 2007 Jan 16;104(3):1027-32.
doi: 10.1073/pnas.0610155104. Epub 2007 Jan 5.

Controlled expression of transgenes introduced by in vivo electroporation

Takahiko Matsuda  1 , Constance L Cepko
Affiliations

Controlled expression of transgenes introduced by in vivo electroporation

Takahiko Matsuda et al. Proc Natl Acad Sci U S A. .

Abstract

In vivo electroporation is a powerful technique for the introduction of genes into organisms. Temporal and spatial regulation of expression of introduced genes, or of RNAi, would further enhance the utility of this method. Here we demonstrate conditional regulation of gene expression from electroporated plasmids in the postnatal rat retina and the embryonic mouse brain. For temporal regulation, Cre/loxP-mediated inducible expression vectors were used in combination with a vector expressing a conditionally active form of Cre recombinase, which is activated by 4-hydroxytamoxifen. Onset of gene expression was regulated by the timing of 4-hydroxytamoxifen administration. For spatial regulation, transgenes were expressed by using promoters specific for rod photoreceptors, bipolar cells, amacrine cells, Müller glia or progenitor cells. Combinations of these constructs will facilitate a variety of experiments, including cell-type-specific gene misexpression, conditional RNAi, and fate mapping of progenitor and precursor cells.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Temporal regulation of gene expression in the retina by using inducible Cre and Flp recombinases. (A) 4OHT-responsive Cre and Flpe (the enhanced form of Flp) recombinases composed of Cre/Flpe and the mutated ligand-binding domain(s) of ER (ERT2) are expressed under the control of the CAG (chicken β-actin promoter with cytomegalovirus enhancer) promoter (10). (B) CALNL-DsRed: Cre/loxP-dependent inducible expression vectors. DsRed is expressed only in the presence of Cre. CAFNF-DsRed: Flp/FRT-dependent inducible expression vectors. DsRed is expressed only in the presence of Flp. (C) A scheme of the experiment. (D–G) P0 rat retinas were coelectroporated with three plasmids: CAG-GFP (transfection control), CALNL-DsRed (D, F, and G), or CAFNF-DsRed (E) as a recombination indicator and CAG-CreERT2 (D), CAG-FlpeERT2 (E), or CAG-ERT2CreERT2 (F and G). The three plasmids were mixed at a mass ratio of 2:3:1 (final concentration, 4.5 μg/μl). The retinas were stimulated without (D–F) or with (G) 4OHT by i.p. injection at P20 and then harvested at P21. Whole-mount preparations of the harvested retinas are shown. The area of electroporation shows that 5–15% of all cells are positive for GFP. (H–K) Sections of the retinas shown in panels D–G. Cell nuclei were stained with DAPI. Approximately 90% (H), 10% (I), 0% (J), and 60% (K) of GFP-positive cells expressed DsRed.
Fig. 2.
Fig. 2.
Gene expression in the retina by using cell-type-specific promoters. P0 rat retinas were coelectroporated with two plasmids: CAG-GFP (transfection control) and retinal cell-type-specific promoter-DsRed. The two plasmids were mixed at a mass ratio of 1:2 (final concentration, 6.0 μg/μl). The retinas were harvested at P20 (A–H and J) or P2 (I), sectioned, and stained with DAPI. Promoters of rhodopsin (A), Nrl (B), Crx (C), Cabp5 (D), Ndrg4 (E), Cralbp (F), clusterin (G), Rax (H), and Hes1 (I and J) were used to express DsRed. Note that DsRed driven by the Rax promoter was not detected at P20, although it was weakly detected at P2–P3.
Fig. 3.
Fig. 3.
Lineage-tracing experiments in the retina by using the Cre/loxP system and cell-type-specific promoters. P0 rat retinas were coelectroporated with three plasmids: CAG-GFP (transfection control), CALNL-DsRed (recombination indicator), and retinal cell-type-specific promoter-Cre. The three plasmids were mixed at a mass ratio of 2:3:1 (final concentration, 6.0 μg/μl). The retinas were harvested at P20, sectioned, and stained with DAPI. Promoters of rhodopsin (A), Nrl (B), Crx (C), Cabp5 (D), Ndrg4 (E), Cralbp (F), clusterin (G), Rax (H), and Hes1 (I) were used to express Cre. Yellow arrowheads indicate the labeled rods. The blue arrowhead indicates the labeled bipolar cell. (In E, there is signal from DsRed in the GFP image due to the very strong signal from DsRed and very weak signal from GFP in amacrine cells.)
Fig. 4.
Fig. 4.
Inducible expression in Müller glial cells. P0 rat retinas were coelectroporated with three plasmids: clusterin promoter-ERT2CreERT2, CALNL-DsRed, and CAG-GFP. The three plasmids were mixed at a mass ratio of 1:3:2 (final concentration, 6.0 μg/μl). (A) A scheme of the experiment. (B) Whole-mount preparation of the transfected retina harvested at P16 without 4OHT stimulation. (C) Whole-mount preparation of the transfected retina stimulated with 4OHT at P14 and harvested at P16. (D) The retina shown in C was sectioned and stained with DAPI. An average of 0.7% of GFP-positive cells expressed DsRed.
Fig. 5.
Fig. 5.
Inducible RNAi in the retina. (A) Diagram of mir30-based RNAi vectors. hU6-mir30: The mir30 expression cassette has the hairpin stem composed of siRNA sense and antisense strands (22 nt each), a loop derived from human miR30 (19 nt), and 125-nt miR30 flanking sequences on both sides of the hairpin. The cassette is expressed under the control of the human U6 promoter. The mir30 primary transcript is processed to generate the mature shRNA. CAG-mir30: The mir30 expression cassette is expressed under the control of the CAG promoter. CALSL-mir30: Cre-dependent inducible shRNA expression vector carrying a floxed transcriptional stop cassette (3xpolyA signal sequences). In the presence of Cre, the mir30 expression cassette is expressed under the control of the CAG promoter. (B–F) Characterization of mir30-based GFP knockdown vectors in 293T cells. (B–D) CAG-GFP and CAG-HcRed were cotransfected into 293T cells without (B) or with (C and D) mir30-based RNAi vectors expressing a shRNA against GFP. (E and F) An inducible RNAi vector, CALSL-mir30(GFPshRNA), was used without (E) or with (F) CAG-Cre. 293T cells were analyzed 48 h after transfection. (G and H) Conditional GFP knockdown in the retina. CAG-GFP, CAG-DsRed, and CALSL-mir30 (GFPshRNA) were coelectroporated without (G) or with (H) the rhodopsin promoter-Cre into P0 rat retinas. The four plasmids were mixed at a mass ratio of 4:4:6:1 (final concentration, 6.0 μg/μl). The retinas were harvested at P20, sectioned, and stained with DAPI.
Fig. 6.
Fig. 6.
Targeted misexpression of Rax and Hes1 in rod photoreceptors. (A and B) Misexpression of Rax and Hes1 in the developing retina using a conventional expression system. CAG-Rax (A) or CAG-Hes1 (B) was coelectroporated with CAG-GFP into P0 rat retinas. The two plasmids were mixed at a mass ratio of 1:1 (final concentration, 3.0 μg/μl). The retinas were harvested at P14, sectioned, and stained with anti-rhodopsin antibody (red) and DAPI (blue). (C) A schematic illustration showing the differentiation process of rod photoreceptors. The expression of Nrl precedes that of rhodopsin. (D and E) Misexpression of Rax and Hes1 in rod photoreceptors by using the rhodopsin promoter-Cre. CALNL-Rax (D) or CALNL-Hes1 (E) was coelectroporated with the rhodopsin promoter-Cre and CALNL-GFP into P0 rat retinas. The three plasmids were mixed at a mass ratio of 2:1:2 (final concentration, 5.0 μg/μl). The retinas were harvested at P20, sectioned, and stained with anti-rhodopsin antibody (red) and DAPI (blue). (F and G) Misexpression of Rax and Hes1 in rod photoreceptors by using the Nrl promoter-Cre. CALNL-Rax (F) or CALNL-Hes1 (G) was coelectroporated with the Nrl promoter-Cre, CALNL-DsRed, and CAG-GFP into P0 rat retinas. The four plasmids were mixed at a mass ratio of 2:1:2:3 (final concentration, 6.5 μg/μl). The retinas were harvested at P20, sectioned, and stained with DAPI (blue).
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