Ординатура / Офтальмология / Английские материалы / Eye, Retina, and Visual System of the Mouse_Chalupa, Williams_2008

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The ability to alter gene expression in vivo has opened a wide range of investigations into gene structure and function. In addition, it has provided a means to visualize specific cells, track cells with a particular history of gene expression, and alter cellular physiology, and it has allowed the production of models of disease. To facilitate the rapid introduction of gene constructs into the visual system, we used electroporation for delivery to the embryonic and early postnatal retina and brain. Plasmids that allow controlled expression of genes or short hairpin RNAs for RNA interference (RNAi) vectors have been developed. These methods open up the possibility of relatively rapid assessment of gene structure and function.

Comparison of gene transfer methods

Electroporation can be compared to other methods of gene delivery in vivo. Several types of viral vectors, including murine oncoretrovirus (Price et al., 1987; Turner and Cepko, 1987), lentivirus (Miyoshi et al., 1997), adenovirus (Bennett et al., 1994; Jomary et al., 1994; Li et al., 1994), and adenoassociated virus (Ali et al., 1996), are in use. There are advantages and disadvantages inherent in the use of viral vectors. The disadvantages are these: (1) It is time-consuming to prepare high-titer virus stocks to achieve efficient gene transfer. (2) Viral vectors have a size limitation for insert DNA.

(3) In general, such vectors do not readily allow introduction of more than two genes into the same cells. (4) Biosafety is a concern for some viral vectors with broad host ranges. However, an advantage that cannot be overlooked for some applications (e.g., lineage analysis) is that integration of the oncoretroviral genome gives stable gene expression throughout all progeny of an infected cell. Electroporated DNA does not integrate efficiently. For some species, transgenic animals can be made by injecting DNA into fertilized eggs. However, producing such strains is invariably slow and often very expensive.

Electroporation bypasses many of the disadvantages cited earlier (Matsuda and Cepko, 2004). This method is faster

and in some cases safer than viral gene transfer methods. The efficiency of electroporation into at least some areas of the developing visual system is quite good, and transgene expression persists for more than a month. There does not appear to be a limitation in terms of the species that can be targeted. Various types of DNA constructs, including RNAi vectors as well as conventional gene expression vectors, are readily introduced into the retina without DNA size limitation. We have even successfully electroporated bacterial artificial chromosome (BAC) constructs, although with more difficulty and low levels of expression (Cherry and Cepko, 2008). More than two different DNA constructs can be introduced at once. We found that at least five plasmids can be coelectroporated into the same cells without a significant reduction in coelectroporation frequency. We have also generated a series of cell type–specific promoter constructs that direct expression to specific retinal cell types and have achieved temporal control using recombinases.

When considering the options for gene transfer, one should carefully evaluate whether transient or stable, clonal or nonclonal gene expression is desirable, as well as the feasibility of applying the various methods to the species under study. In addition, it is important to recognize that electroporation, like most viral transduction methods, does not transduce 100% of cells in a targeted area. Transgenic animals can provide more uniform transduction, and if this is required, then electroporation and viral methods cannot be used.

Method of electroporation

Retina in Vivo The basic strategy for in vivo electroporation into the retinas of newborn mouse and rat pups is to inject DNA into the subretinal space between the retina and retinal pigment epithelium (RPE) (figure 51.1A). Electrodes are then placed on the heads of the pups and electric pulses are applied to the eyes (figure 51.2). The DNA constructs are transduced into the scleral side of the retina, where undiffentiated mitotic and newly postmitotic cells

exist (figure 51.3). Because DNAs are preferentially transduced into undifferentiated progenitor/precursor cells, only late-born cell types (rod, bipolar, Müller glia, and a subset of amacrine cells) are labeled by electroporation at P0 (figures 51.4 and 51.5). It is not clear whether electroporation

Figure 51.1 Strategy for in vivo electroporation A, Electroporation to the scleral (RPE) side of the retina. B, Electroporation to the vitreal side of the retina. See color plate 50.

into progenitor/precursor cells is more effective than into mature neurons because of an inherent difference in cell types or whether this is simply due to the location of progenitor/precursor cells adjacent to the DNA injection site, the subretinal space. In addition to this strategy, it is also theoretically possible to transfect DNAs from the vitreous side of the retina by injecting DNAs into the vitreous chamber, and applying electric pulses in the direction opposite to that used for subretinal injections (see figure 51.1B). Indeed, other groups reported that DNA constructs could be transduced to ganglion cells, which line the surface of the retina facing the vitreous body, by in vivo electroporation using this strategy (Dezawa et al., 2002; Huberman et al., 2005; Kachi et al., 2005). However, our data show that the transfection efficiency of the vitreal side (ganglion cells) of the neonatal retina, as well as of the adult retina, is much lower than of the scleral side (progenitor/precursor cells) of the neonatal retina (see figure 51.8). Again, it is not clear why this injection site results in less successful transduction. It could result from physiological differences between ganglion cells/displaced amacrine cells and progenitor/ precursor cells; for example, it could be the case that DNA plasmids may not be readily transcribed in these neurons. Alternatively, or in addition, it could result from such things as access of the electroporated plasmids to the nuclear

Figure 51.3 Whole-mount preparation of rat retina in vivo electroporated at P0 with CAG-GFP (Matsuda and Cepko, 2004), a GFP expression vector driven by the CAG (chicken β-actin pro-

moter with cytomegalovirus enhancer) promoter and harvested at P21. Images are from the scleral side. Bright-field (A), GFP (B), and merged (C ) images are shown. See color plate 52.

Figure 51.4 In vivo electroporated retina (P0 electroporation, section). Rat retinas were in vivo electroporated with CAG-GFP at P0 and harvested at P2 (top panel) or P20 (bottom panel). At P2, most of the GFP-positive cells have the morphology of progenitor/precursor cells, suggesting that DNAs are preferentially transfected to progenitor/precursor cells. Retinogenesis is completed within the

compartment of the neurons. For example, electroporated plasmids may enter a cellular compartment, such as axons, Müller glial endfeet, or blood vessels, or even get trapped in basement membrane rather than go directly into the ganglion cell cytoplasm.

To deliver DNAs into early-born cell types (cone, horizontal, ganglion, and amacrine cells) whose progenitor/ precursor cells exist primarily in the embryonic retina, one needs to electroporate DNAs into embryonic retinas (see figure 51.5). Two approaches are used for in vivo (in utero) electroporation into embryonic retinas. One utilizes ultrasound to guide delivery of the plasmid DNA to the subretinal space, or early optic vesicle, such as in murine embryos at E9.5. One can also deliver plasmid DNA to pups in utero without ultrasound, from about E13 for mouse or E14 for rat. This approach involves a learning period during which

first 2 weeks after birth. At P20, GFP is observed in four differentiated cell types: rod photoreceptors, bipolar cells, amacrine cells, and Müller glia. Early-born cell types (cone, horizontal, and ganglion cells) are not labeled by P0 electroporation. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; OS, outer segment; VZ, ventricular zone. See color plate 53.

the practitioner becomes familiar with the landmarks of the embryonic structures as seen through the uterine wall (Turner et al., 1990). Delivery to the embryonic tissue again appears to result in uptake of the DNA primarily by cycling cells or newborn neurons. Because embryonic cells tend to undergo more rounds of cell division than postnatal progenitor cells, the DNA is diluted out more rapidly. Retention of the plasmid in the neurons that are generated soon after electroporation is apparent, because those are the cells that are most strongly marked; an example is the amacrine cells and cone photoreceptors following delivery at E14 in the mouse (figure 51.6).

Retina in Vitro In organ cultures of embryonic or neonatal retina, progenitor cells differentiate into neurons and glia and form three layers, mimicking normal development.

Figure 51.5 Timing of electroporation and labeled cell types. Birth order of retinal cells in the mouse retina is shown. As DNAs are preferentially transfected into undifferentiated progenitor/precursor cells by electroporation, electroporation at P0 labels only late-born cell types (rod, bipolar, Müller glia, and a subset of ama-

Figure 51.6 In vivo electroporated retina (E14 electroporation, section). Mouse embryonic retinas were electroporated with UBGFP (Matsuda and Cepko, 2004), a GFP expression vector driven by the human ubiquitin promoter, at E14 in utero, and harvested at P20. Early-born cell types (cone, amacrine, horizontal, and ganglion cells) are clearly labeled with GFP, while late-born cell types (bipolar and Müller glial cells), which are generated from E14

crine cells), which are generated from P0 retinal progenitors. On the other hand, electroporation at E14 can label early-born cell types (cone, horizontal, ganglion, and amacrine cells), which are generated from E14 retinal progenitors.

RPCs after several rounds of cell division, are poorly labeled. This is probably due to dilution of introduced plasmids. Red arrowheads indicate the labeled cone photoreceptors. Yellow arrowhead indicates the labeled horizontal cell. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; OS, outer segment. See color plate 54.

Taking advantage of this propensity, we also developed a system to electroporate DNAs into isolated retinas (in vitro electroporation; see Matsuda and Cepko, 2004) using a micro-electroporation chamber (figures 51.7 and 51.8). Electroporated retinas are cultured for a few days to weeks.

Compared with in vivo electroporation, in vitro electroporation has several advantages. First, in vitro electroporation is easier and less skill dependent than in vivo electroporation. All retinas subjected to electroporation become GFP (RFP) positive when GFP (RFP) expression

vectors are used. Second, it is relatively easy to handle a large number of retinas in a day. Third, in vitro electroporation can be easily applied not only to postnatal retinas, but also to embryonic retinas, to which in vivo electroporation (in utero electroporation) is more difficult to apply. Fourth, realtime monitoring of GFP (RFP)-transduced cells is possible under a fluorescent microscope. However, in vitro electroporation has several disadvantages inherent to organ culture. First, the morphology of cultured retina is frequently poor, and photoreceptor outer segments are poorly formed. Second, it is hard to culture retinas for a long period. In our

Figure 51.8 In vitro electroporated retinal explant (whole mount). Mouse retinas of P0 CD1 (A and B), adult CD1 (C and D), or adult Swiss Webster mice with a retinal degeneration mutation (E and F) were in vitro electroporated with CAG-GFP from the scleral side (A, C, and E) or from the vitreal side (B, D, and F) and cultured for

5 days. Images A, C, and E are from the scleral side and images B, D, and F are from the vitreal side. Note that only the scleral side of developing retina or of degenerated retina is highly transfectable. In E, most of the GFP-positive cells are Müller glial cells. See color plate 56.

experience, retinas tend to become unhealthy when cultured for more than 2 weeks.

Brain in Vivo The delivery of plasmid DNAs to the brain has been used by several groups previously (FukuchiShimogori and Grove, 2001; Saito and Nakatsuji, 2001; Tabata and Nakajima, 2001). The same issues discussed earlier pertain here as well, such as dilution of DNA following early delivery (Shimogori et al., 2004). Marking of visual cortex has been achieved (Akaneya et al., 2005; Huberman et al., 2005). It is likely that most areas adjacent to a ventricle will allow successful plasmid uptake and expression following electroporation. Ventricular zones are adjacent to the ventricular lumina, into which DNA can be readily injected, and they are also the area where progenitor cells reside. As discussed earlier for the retina, it appears that these two criteria offer the ideal circumstances for successful electroporation. Injection into tissue rather than into a lumen does not result in effective electroporation, likely because only the DNA that remains in the needle track is available for uptake. In addition, it is unclear whether the neurons, which are more mature in developing tissue, will successfully take up and express such plasmids.

Controlling expression following electroporation

One advantage to using transgenic animals rather than viral vectors or even (often) electroporation is the ability to use regulatable promoters and recombinases to control gene expression. We have created a series of plasmids to realize these advantages for electroporated genes, or RNAi contstructs (Matsuda and Cepko, 2007).

Temporal Regulation Conditionally active Cre recombinases are composed of Cre and the mutated ligand binding domains of the human estrogen receptor (ERT2). They are activated by 4-hydroxytamoxifen (4-OHT) and have been used to temporally control gene expression in transgenic mice (Branda and Dymecki, 2004). We tested the 4-OHT- responsible Cre recombinases, CreERT2 (single fusion; Feil et al., 1997) and ERT2CreERT2 (double fusion; Casanova et al., 2002), in vivo in the rat retina. These recombinases were expressed under control of the ubiquitous CAG promoter (Niwa et al., 1991) (figure 51.9A). A Cre-dependent expression vector (Kanegae et al., 1995) containing the CAG promoter, a floxed stop cassette, and a reporter gene (DsRed) was used as a recombination indicator (termed CALNLDsRed; figure 51.9B). When P0 rat retinas were coelectroporated with CAG-CreERT2, CALNL-DsRed (recombination indicator), and CAG-GFP (transfection control) and harvested at P21, very high background recombination (DsRed expression) was detected even without 4-OHT stimulation (figure 51.9D). ERT2Cre (N-terminal ERT2 fusion)

also had very high background recombination activity. In contrast, ERT2CreERT2 double fusion had no detectable recombination activity without 4-OHT (figure 51.9E). When 4-OHT was injected intraperitoneally into the transfected rats at P20, an induction of DsRed expression was clearly detected 24 hours after 4-OHT administration (figure 51.9F). Similar results were observed when CreERT2 and ERT2CreERT2 were transfected into 293 T cells or E14.5 mouse brain. These results indicate that at least for in vivo electroporation studies, ERT2CreERT2, but not CreERT2 (ERT2Cre), can lead to tight regulation of the onset of transgene expression.

It is not clear why the double ERT2 domain construct was required for tight regulation of recombinase activity. The heat shock protein 90 (Hsp90) interacts with the ER domain in the cytosol and thereby prevents the translocation of CreER fusion protein to the nucleus where DNA recombination occurs (Picard, 1994). The double fusion may have a higher affinity for Hsp90 to form a tighter complex. Alternatively, the ERT2CreERT2 fusion may have less activity due to the double fusion, and thus less background activity. It is also possible that degradation of CreERT2 (ERT2Cre) results in generation of “active Cre” lacking the regulatory domain, while ERT2CreERT2 is still inactive even after losing one regulatory domain.

Cell Type–Specific Regulation Using Specific Promoters To restrict transgene expression to specified cell types in the retina, several retinal cell type–specific promoters were obtained using the literature to guide construction or were developed in our laboratory. Regulatory sequences for rhodopsin (expressed in rods; Zack et al., 1991), Nrl (expressed in rods; Swaroop et al., 1992), Crx (expressed in photoreceptors and weakly in bipolars; Chen et al., 1997; Furukawa et al., 1997b), calcium-binding protein 5 (Cabp5, expressed in subsets of bipolar cells; Haeseleer et al., 2000), N-myc downstream-regulated gene 4 (Ndrg4, expressed in amacrines; Punzo and Cepko, 2008), cellular retinaldehyde-binding protein (Cralbp, expressed in Müller glia; Kennedy et al., 1998), clusterin (expressed in Müller glia; Blackshaw et al., 2004), Rax (expressed in progenitors and Müller glia; Furukawa et al., 1997a; Mathers et al., 1997), and Hes1 (expressed in progenitors and Müller glia; Tomita et al., 1996) were characterized for this purpose. When these promoters were used to express DsRed, DsRed was detected only in specific cell types in the retina (figure 51.10 and table 51.1). Using fluorescent protein variants (CFP, YFP, and DsRed) as reporters, we could visualize different cell types simultaneously (figure 51.11).

The cell type–specific promoters were also used to regulate expression of Cre recombinase (figure 51.12). This type of construct was coelectroporated with CALNL-DsRed, a recombination indicator. Following the action of Cre, DsRed

Figure 51.9 Temporal regulation of gene expression in the retina using inducible Cre recombinases. A, CAG-CreERT2: Fusion protein (CreERT2) between Cre recombinase and the mutated ligand-binding domain (ERT2) of the human estrogen receptor is expressed under the control of the CAG promoter. CAG-ERT2 CreERT2: Fusion protein (ERT2CreERT2) composed of Cre and two ERT2 domains is expressed under the control of the CAG promoter. CreERT2 and ERT2CreERT2 are conditionally activated in response to 4-OHT. B, CALNL-DsRed: Cre/loxP-dependent inducible expression vector. DsRed is expressed only in the presence of Cre. C, A scheme of the experiment. D–I, P0 rat retinas were coelectroporated with three plasmids: CAG-GFP (transfection control),

would be expressed from the ubiquitous CAG promoter. In all cases, DsRed expression levels in the retina were higher when DsRed was expressed from the CAG promoter rather than any of the cell type–specific promoters. The rhodopsin promoter-Cre construct specifically induced the expression of DsRed in rods. Similarly, the Nrl promoter-Cre construct led to the expression of DsRed only in rods, indicating that both promoters are restricted to rods and are not even tran-

CALNL-DsRed (recombination indicator), and CAG-CreERT2 (D) or CAG-ERT2CreERT2 (E and F). The retinas were stimulated without 4-OHT (D and E) or with 4-OHT (F) by IP injection at P20 and then harvested at P21. Whole-mount preparations of the harvested retinas are shown. G–I, Sections of the retinas shown in D–F. Cell nuclei were stained with DAPI. When CreERT2 was used, significant background recombination was observed even in the absence of 4-OHT. On the other hand, ERT2CreERT2 had no detectable basal activity in the absence of 4-OHT. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. See color plate 57.

siently active in other cell types, including multipotent progenitors. Interestingly, when the Cabp5 promoter-Cre construct was used, a subset of rods, as well as bipolars, was labeled with DsRed. The ratio of the number of DsRedpositive rods to that of DsRed-positive bipolars was approximately 1 : 1. This might suggest that the Cabp5 promoter is active in the progenitors that produce rod and bipolar cells. The Ndrg4 promoter-Cre construct induced the expression