Self-inactivating lentiviruses: Versatile vectors for quantitative transduction of cerebellar granule neurons and their progenitors

Journal of Neuroscience Methods 149 (2005) 144–153

Self-inactivating lentiviruses: Versatile vectors for quantitative transduction of cerebellar granule neurons and their progenitors Wei Wang a , Qiang Qu b , Frances I. Smith b , Daniel L. Kilpatrick a,∗ a

Department of Molecular and Cellular Physiology, University of Massachusetts Medical School, Basic Science Building, BSB S4-139, 55 Lake Avenue North, Worcester, MA 01655, USA b Shriver Center for Mental Retardation, Waltham, MA 02452, USA Received 18 January 2005; accepted 12 May 2005

Abstract Cerebellar granule neurons (CGNs) undergo a well-defined, intrinsic differentiation program that is recapitulated in vitro. Thus, homogeneous cultures of CGNs provide an excellent opportunity to define the mechanisms underlying their development. The ability to alter endogenous gene expression in CGNs on a population-wide basis would greatly facilitate the elucidation of these events. In the present study, we show that self-inactivating lentiviruses efficiently infect both dividing progenitors and post-mitotic CGN cultures in a quantitative manner without altering their cellular properties. The time course for protein expression was biphasic for both types of cultures, with the first peak occurring during the initial infection period. Thus, lentiviruses can express proteins in CGNs both acutely and on a long-term basis to study developmental and other processes continuously over an extended time period. These vectors also infected CGNs in cerebellar slice preparations. In addition, lentiviruses harboring a transgene for the mouse GABAA receptor ␣6 subunit promoter recapitulated the differentiation-dependent expression of this gene in CGN cultures. Self-inactivating lentiviruses are extremely versatile vectors that offer important advantages for studies of protein function and gene regulation. The ability to alter protein function on a global scale in CGN cultures permits biochemical assessment of its impact on mRNA and protein populations, as well as on protein–protein and protein–DNA interactions. Further, integrated lentiviruses can be used to study chromatin-dependent promoter regulation and transcription factor interactions in CGNs over time in a facile manner. © 2005 Elsevier B.V. All rights reserved. Keywords: Viral transduction; Differentiation; Chromatin; Promotor

1. Introduction Neuronal development is a complex process that requires a highly coordinated program of gene expression, and a critical question is how this program is controlled in a spatiotemporal manner. Cerebellar granule neurons (CGNs) undergo a well-defined series of differentiation events that serve as a useful model for neuronal maturation (Hatten et al., 1997). During early postnatal development, proliferation of CGN progenitors occurs within the external germinal layer (EGL). Progenitors then exit the cell cycle and initiate differentiation by extending bipolar axons in the premigratory zone. They ∗

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0165-0270/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jneumeth.2005.05.019

then extend a third, vertical process and migrate through the molecular and Purkinje layers to form the internal granule layer (IGL). Maturation of CGNs is then completed within the IGL, including dendrite formation and synaptogenesis. Interactions with neighboring cells are critical for normal development of CGNs, including regulation of progenitor proliferation by Purkinje cell-derived sonic hedgehog (shh) (Wechsler Reya and Scott, 1999). However, many aspects of CGN maturation are intrinsically determined (Carletti et al., 2002; Yacubova and Komuro, 2002; Yamasaki et al., 2001), including the expression of differentiation-related genes (Gao and Fritschy, 1995; Lin and Bulleit, 1996). CGNs are very abundant neurons and can be prepared and cultured as essentially homogeneous cells (Hatten, 1985). These preparations consist mainly of CGN progenitors (CGNPs) derived from

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the EGL (Hatten, 1985; Raetzman and Siegel, 1999). Isolated CGNPs can be cultured as proliferating cells (Gao et al., 1991; Wechsler Reya and Scott, 1999) or as post-mitotic neurons that recapitulate a fairly synchronous, intrinsic differentiation program (Lin and Bulleit, 1996; Raetzman and Siegel, 1999). This versatility permits mechanistic analysis of both early and late stages of CGN differentiation. Further, their maturation also can be studied in an intact cellular context using cerebellar slices (Komuro and Rakic, 1992). Thus, CGNs provide an excellent system in which to explore key questions of neuronal maturation, including the proper timing of successive differentiation events. The ability to perturb the expression and function of regulatory proteins is a critical approach in elucidating the mechanisms underlying cell differentiation. Plasmid transfection has been used extensively to express proteins of interest in CGNs and other neurons (Ango et al., 1999; Wellmann et al., 1999; Yang et al., 2004). A limitation with this approach is its relatively low efficiency, which necessitates the analysis of individual transfected cells. This precludes molecular studies designed to screen for genes lying downstream of a protein of interest, or biochemical studies of protein–protein and DNA–protein interactions. In the present study, we have examined the utility of lentiviruses for effecting quantitative protein expression in CGNs. These vectors offer important advantages over other viral systems, including ease of generation, relatively high titer, low cytoxicity, broad host expression and the ability to infect both dividing and nondividing cells (Trono, 2000). Their integration into the host genome also provides for persistent, stable expression that is highly useful for studying developmental events over time as well as chromatin-dependent transcriptional regulation of gene promoters. Here, we demonstrate that lentiviruses can quantitatively express proteins in CGN cultures, permitting a more expanded approach to studying the neuronal differentiation program. We also describe their versatility as transgene vectors for analyzing gene promoters in differentiating primary neurons.

2. Materials and methods 2.1. Cell & cerebellar slice cultures Mouse CGNs were prepared from postnatal 6 (P6) CD1 pups as previously described (Hatten, 1985). Briefly, dissected cerebella were digested with 1% trypsin, 1 mg/ml DNase (Sigma, St. Louis, MO) in calcium-magnesium free PBS (pH 7.4) at room temperature for 3 min. CGNs were prepared by mechanical trituration and enriched by PercollTM (Sigma) gradient centrifugation. The granule cell fraction was further purified by pre-plating the cells on poly-dlysine-coated Petri dishes at 35 ◦ C for 1 h. Purified cells were plated on plastic dishes or on glass coverslips coated with 1 mg/ml poly-d-lysine at a density of 2 × 106 ml−1 in NeurobasalTM medium containing B-27 serum-free sup-

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plements, 2 mM l-glutamine, 100 U/ml penicillin/100 ␮g/ml streptomycin (Invitrogen, Grand Island, NY) and 0.45% glucose at 37 ◦ C/5% CO2 . For CGNPs, cells were cultured in medium containing 3 ␮g/ml sonic hedgehog (R&D System Inc., MN). In certain experiments, shh was later removed to induce differentiation. Mouse cortical neurons were prepared from E18 CD1 pups. Dissected cortices were digested with 0.05% trypsin at room temperature for 2 min. After mechanical trituration, the cells were plated on plastic dishes coated with 100 ␮g/ml poly-d-lysine at a density of 2 × 106 ml−1 in the same NeurobasalTM /B27 medium used for CGNs. Human embryonic kidney 293T and human choriocarcinoma JEG3 cells were obtained from American Type Culture Collection (Rockville, MD). The cells were grown in Dulbecco’s modified Eagle’s medium (Sigma) containing 10% heat-inactivated fetal bovine serum (Invitrogen, Grand Island, NY). Cerebellar slice cultures were prepared as previously described (Baboval et al., 2000). Briefly, 250 ␮m sections were cut from P6 cerebella embeded in 5% agrose, incubated in MEM medium (Sigma) containing 10% FBS, 2 mM lglutamine, 100 U/ml penicillin/100 ␮g/ml streptomycin and 0.45% glucose at 35 ◦ C/5% CO2 , and then placed onto Transwell membrane inserts (Costar, Cambridge, MA). The slices were cultured in the same NeurobasalTM /B27 medium used for CGNs. 2.2. Lentiviral plasmids, viral production and transduction of cells Self-inactivating lentiviral vectors pHR -CMV LacZSin18 (CMV-␤Gal) (Zufferey et al., 1998), pHR -cPPTCMV-HA-EnR-W-Sin18 and pHR -cPPT-CMV-HA-NFIBW-Sin18 (Wang et al., 2004) were previously described. A lentiviral vector containing the promoter for the mouse ␣6 subunit of the GABAA receptor (GABRA6) fused to downstream IRES and luciferase reporter sequences (Wang et al., 2004) was used for promoter studies. VSV-G-pseudotyped lentiviral particles were generated by transient co-transfection of the vector construct (15 ␮g), the packaging construct pCMV
8.91 (10 ␮g) and the pMD.G VSV-G viral envelope expression vector (5 ␮g) into 293T cells using the Calcium Phosphate ProFection Mammalian Transfection System (Promega, Madison, WI) according to the manufacturer’s protocol. The viruses were harvested 48 h later, passed through a 0.45 ␮m filter and further concentrated by ultracentrifugation as previously described (Reiser, 2000). The titers for viruses expressing ␤galactosidase and (hemagglutinin-) HA-tagged NFI-B were determined by transducing JEG3 cells (105 cells) with a viral serial dilution. The dilution resulting in 50–100 cells stained using either X-Gal (Fisher Biotech, Pittsburgh, PA), or HA antibodies (Cell Signaling, Beverly, MA) was determined to calculate the titer (Condit, 2001). The viral stocks for promoter constructs were titered by measurement of p24 levels

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using an HIV-1 p24 ELISA kit (ZeptoMetrix Corporation, Buffalo, NY). Viral transductions were typically performed in 48-well plates or 8-well chamber slides in a total volume of 200 ␮l for 8 h. For slice culture infection, 5 ␮l of viral-containing medium (∼3 × 105 infection units) was added to the surface of each slice and left for 6 h followed by washing and addition of fresh culture medium. Immunofluorescence for HA as well as NeuN was performed 48 h post-infection. 2.3. Enzyme and immunofluorescence assays ␤-galactosidase expression was measured by X-Gal staining of cell cultures as previously described (Wang et al., 2004). Luciferase or ␤-galactosidase activities assays were determined in 10 ␮l of cell extracts using either the luciferase assay system (Promega, Madison, WI) or Galacto-LightTM & Galacto-Light PlusTM Systems (Applied Biosystem, MA), respectively. Data points were performed in triplicate, and the results of at least three independent experiments were averaged to determine the mean ± S.D. for luciferase activity. Student’s t-test was used for statistical analysis. For immunofluorescence, cells or slices transduced with virus were fixed with 4% paraformaldehyde in PBS for 30 min at room temperature. They were blocked with 10% normal goat serum for 30 min at room temperature and then treated with primary antibody. The following antibodies were used: anti-NeuN (1:800, Chemicon), anti-GABRA6 (1:400, Chemicon), anti-HA (1:400, Cell Signaling). CY-3 or FITCconjugated secondary antibodies were used to detect binding of primary antibodies. For 5-bromo-2 -deoxy-uridine (BrdU) incorporation assays, cells were pulse labeled for 3 h and incorporation of BrdU was analyzed using a detection kit (Roche, Indianapolis, IN) according to the manufacturer’s instructions.

3. Results 3.1. Self-inactivating lentiviral vectors efficiently transduce primary CGN cultures We examined the utility of lentiviruses in transducing shh-treated progenitors as well as early post-mitotic CGN cultures at varying MOIs on day 1 in vitro (1 DIV). A self-inactivating lentivirus was used that expresses ␤galactosidase under the control of the CMV promoter. This vector contains both woodchuck and polypyrimidine tract post-transcriptional regulatory elements that augment transgene expression (Barry et al., 2001; Zufferey et al., 1998, 1999). At MOI 0.5, ∼20% of post-mitotic CGNs and ∼35% of CGNPs were infected, while at MOI 2 70–85% of cells expressed ␤-galactosidase (Fig. 1A and B). No significant cell toxicity was observed for either cell culture preparation between MOI 0.5–2.0 based on cell viability assays (data not shown). Thus, transduction efficiencies for CGNP and

CGN cultures infected on 1 DIV were similar, especially at MOI ≥ 1, and a large majority of cells were infected at MOI 2 in both cases. The ability to transduce proliferating and differentiating cells at different time points during culture can be quite useful for addressing stage-dependent protein function. We therefore investigated infection by self-inactivating lentiviruses on different days in culture. CGNPs showed no significant differences in transduction frequencies when infected with CMV␤Gal virus on 1 or 3 DIV (data not shown). However, postmitotic CGNs exhibited a dramatic decline in ␤-galactosidase activity when infected on successive days (Fig. 2A). Thus, transducibility of post-mitotic CGNs declines dramatically with time in culture. Inclusion of polybrene (2–8 ␮g/ml), which is frequently used to increase lentiviral infection (Dull et al., 1998), on 3 DIV was unsuitable due to substantial cell death 48 h post-infection (data not shown). The selective decline in viral transduction with time in culture for post-mitotic CGNs may have reflected their decreased cell proliferation relative to shh-treated cells. To address this, bromodeoxyuridine (BrdU) incorporation was examined between 1 and 3 DIV for both culture formats. On 1 DIV, ∼45% of Shh-treated cells and ∼20% of untreated cells were labeled (Fig. 2B). Over the next 2 days, essentially no BrdU incorporation was observed in differentiating cultures while levels in shh-treated cultures remained high (47 and 53%, respectively). Thus, while cell proliferation declined in differentiating CGNs between 1 and 3 DIV, it did not correlate closely with lentiviral transduction. For example, transduction of these cells on 2 DIV was several-fold higher than on 3 DIV (Fig. 2A). 3.2. Expression patterns for lentiviral-transduced proteins in CGN cultures We next examined the time course of protein expression in CGNP and CGN cultures infected with self-inactivating lentiviral vectors. Cells were transduced on 1 DIV and then assayed on subsequent days for ␤-galactosidase activity. Interestingly, a bimodal expression pattern was observed in both progenitor and CGN cultures (Fig. 3). An initial peak was observed at 6 h post-infection, after which activity declined before increasing to even higher levels between 48 and 72 h after adding virus. Thus, lentivirus-mediated expression occurs both acutely and long-term in CGN cultures. 3.3. Effects of lentivirus infection on granule cell proliferation and differentiation For viral infection to be useful in analyzing function, it must not significantly alter the cellular properties under investigation. We therefore examined the impact of selfinactivating lentivirus on CGN proliferation and differentiation. When CGNPs were infected with CMV-␤Gal virus, no difference in BrdU incorporation was observed between virus- and mock-infected cells (Fig. 4A). NeuN is a nuclear

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Fig. 1. ␤-galactosidase expression in CGNs and CGN progenitors transduced with pHR -CMV-LacZ-SIN18. (A) Cultures were infected on 1 DIV at the indicated MOI. After 72 h, cells were stained with X-Gal. (B) Quantitation of LacZ activities on 3 DIV following infection on 1 DIV.

protein that serves as a marker for differentiating CGNs within the mouse cerebellum (Weyer and Schilling, 2003), and is initially expressed soon after CGNs exit the cell cycle. We therefore monitored NeuN expression to determine whether lentiviral infection affected early granule cell differentiation. CGNPs were infected with CMV-␤Gal virus on 1 DIV and shh was removed 48 h post-infection to trigger cell differentiation. Again, no difference was seen in NeuN stain-

ing between virus-infected and control cells (Fig. 4B). Thus, lentivirus infection did not alter the ability of CGNPs to either divide or initiate terminal differentiation. GABRA6 is highly enriched in CGNs and its expression initiates late in their differentiation, after migration to the IGL is completed and dendritogenesis begins (Zheng et al., 1993). This process is intrinsically controlled, occurring with a delayed time course in purified cultures of CGNs after 4–6

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infected on 1 DIV at MOI 2 and cultured to 6 DIV. RTPCR revealed no differences in GABRA6 transcript levels between control cells and those infected with CMV-␤Gal virus (data not shown). Further, immunofluorescence demonstrated that expression of GABRA6 protein was unaltered in virus-infected cells (Fig. 4C). Thus, lentiviral infection has no significant effect on either early or late differentiation markers in CGNs. 3.4. Transduction of cortical neurons We also examined the ability of lentivirus to express proteins in neuronal cell types other than CGNs. Cortical neurons were prepared from E18 mice and infected with CMV-␤Gal virus on 1 DIV, as for CGN cultures. LacZ expression on 6 DIV for cortical neurons was somewhat higher than that observed in CGN cultures (Fig. 5A). Thus, VSV-G pseudotyped lentiviral vectors are effective at transducing both mouse cortical neurons and CGNs.

Fig. 2. Lentiviral transduction of CGNs declines with time in culture. (A) CGNs were infected with pHR -CMV-LacZ-SIN18 at MOI of 0.5 on 1, 2 or 3 DIV. ␤-Galactosidase activity was analyzed 72 h following infection. (B) Proliferation by CGN and CGNP cultures was determined between 1 and 3 DIV using BrdU incorporation.

days in vitro (Lin and Bulleit, 1996). We therefore examined GABRA6 expression in lentivirus-infected post-mitotic CGN cultures as a measure of viral impact on later stages of their terminal differentiation. Post-mitotic CGNs were

3.5. Lentiviruses as transgenic promoter vectors in primary neurons The analysis of gene promoter activity and its regulation is an important approach in studying cell differentiation and gene function. Transfection of promoter plasmids is often difficult in primary post-mitotic neurons, and the regulatory information provided can be limited by the transient and episomal nature of the transfected promoter DNA. The efficient and stable integration of lentiviral transgenes into the DNA of post-mitotic neurons can potentially overcome these limitations. We therefore examined the expression of a lentiviral vector containing the mouse GABRA6 promoter (Bahn et al., 1997) linked to the luciferase reporter in postmitotic CGNs. To minimize potential promoter interference by viral sequences, a lentiviral vector was used that lacked WPRE and cPPT post-transcriptional elements. For example, upstream cPPT elements can influence transcription from a downstream promoter (Han and Zhang, 2002). As noted earlier, the GABRA6 gene is expressed with a delayed time course in vivo and in CGN cultures, becoming robust between 4 and 6 DIV (Lin and Bulleit, 1996). The GABRA6 promoter lentivirus efficiently expressed luciferase activity that increased ∼2.5-fold between 4 and 6 DIV (Fig. 5B), mirroring the expression pattern of the endogenous gene. Thus, lentiviral vectors are well-suited for studying promoter activity in primary neurons in a physiological chromatin context, including differentiation-dependent regulation. 3.6. Gene transfer in cerebellar slices

Fig. 3. Kinetics of ␤-galactosidase expression following transduction with pHR -CMV-LacZ-SIN18. CGNs (A) and CGN progenitors (B) were infected on 1 DIV at MOI 0.5. ␤-Galactosidase activity was determined at the indicated times.

Organotypic slice cultures of neuronal tissue preserves many of the cellular relationships present in vivo and permit analyses of more complex behavior such as cellular migration (Roberts et al., 1993). We investigated the ability of

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Fig. 4. Characterization of lentiviral-infected CGNs. (A) CGN progenitors were infected with LacZ-expressing virus at MOI 2. Upper panels, the proliferation of viral infected CGN progenitors was compared with mock infected cultures using BrdU incorporation assays. The percentage of BrdU-positive cells is indicated. Lower panels, phase contrast images of the same fields. (B) CGN progenitors were infected with LacZ-expressing virus at MOI 2. Forty-eight hours after infection, shh was removed and cells were fixed for NeuN staining at 72 h post-infection (upper panels). The percentage of NeuN-positive cells is indicated. Lower panels, phase contrast images of the same fields. (C) CGNs were infected on 1 DIV with pHR -cPPT-CMV-HA-EnR-W-Sin18 virus (MOI 1) that expresses HA-tagged Drosophila engrailed. On 7 DIV, cells were fixed and stained with anti-HA and anti-GABRA6 antibodies.

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the IGL, numerous HA-expressing CGNs were identified that also expressed the granule marker NeuN (Fig. 6). Thus, lentiviral vectors also transduce granule neurons in cerebellar slices, and can be used to study more complex properties of these cells in their native environment.

4. Discussion

Fig. 5. Lentiviral vectors in studies of different neuronal subtypes and of differentiation-dependent promoter regulation. (A) Post-mitotic CGNs and cortical neurons were infected on 1 DIV with pHR -CMV-LacZ-SIN18 lentivirus and then assayed on 6 DIV. (B) Post-mitotic CGNs were infected on 1 DIV with a lentiviral vector expressing the mouse GABRA6 promoter and were harvested and assayed on either 4 or 6 DIV for luciferase activity. ␤Galactosidase activity from co-infected pHR -CMV-LacZ-SIN18 virus was used for normalization.

self-inactivating lentivirus to transduce granule neurons in cerebellar slices prepared from P6 mice. A lentivirus expressing an HA-tagged version of the transcription factor NF-IB2 was used to study co-localization of viral expressed protein and the CGN marker NeuN within the same nucleus. Within

Functional perturbation of regulatory proteins in neuronal cells is critical to understanding their development and physiology. The inability to stably express proteins in a high percentage of cells in a neuronal population using plasmid transfection precludes its use beyond the analysis of individual cells. Various viral vectors have been used to overcome the relative refractoriness of neurons to transient transfection, including adenovirus, sindbis virus, and murine retroviruses (Ehrengruber, 2002; Kageyama et al., 2003; Slack and Miller, 1996). However, these vectors exhibit properties that typically limit their use in neuronal studies, including alteration of cell viability or cellular function (Cregan et al., 2000; Kim et al., 2004; Sato et al., 2004), low viral titers and the inability to infect post-mitotic cells as seen with murine retroviruses. Efficient transduction of neurons has been previously reported using herpes simplex, Semliki forest, and adeno-associated viruses (Sato et al., 2004; Wang et al., 2002; Wu et al., 2002). However, these vectors also are limited by cytotoxicity effects and transient expression, or in the case of adeno-associated virus, relatively low insert size capacity (Monville et al., 2004; Slack and Miller, 1996). Self-inactivating lentiviruses

Fig. 6. Transduction of organotypic slice cultures with lentivirus. Cerebellar slices prepared from P6 mice were infected on 0 DIVwith lentivirus expressing HA-tagged NFI-B. Slices were stained for HA and NeuN 48 h after infection. Sections are shown at two different magnifications. HA-expressing CGNs are indicated by arrowheads.

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overcome these difficulties: they stably transduce neurons without significant alteration in their cellular properties and tolerate relatively large transgenes (inserts >18 kb have been reported (Kumar et al., 2001)). Lentiviruses also can be prepared with a much higher titer (∼108 ifu/ml) than generally obtainable with murine retroviruses, for example. The quantitative extent of transgene expression obtainable with lentivirus makes it possible to perturb gene expression and protein function en masse within CGN cultures. Thus, the effects of exogenous proteins on gene expression can be monitored biochemically using extracted mRNAs, as recently demonstrated for NFI-dependent regulation of the GABRA6 gene in CGNs (Wang et al., 2004). Further, microarray approaches can be used to identify novel downstream effects on gene expression induced by a transgene. These vectors also can be used to study alterations at the protein level, including post-translational status, protein–protein and protein–DNA interactions that are typically difficult to analyze histologically. For example, it is possible to study the direct interaction of an exogenously expressed nuclear protein with specific gene promoters in primary neurons using chromatin immunoprecipitation. Similarly, transgeneinduced alterations of in vivo promoter binding by endogenous factors also can be directly assessed using these vectors. Another advantage of lentiviral vectors is the ability to infect and study the differentiation of both neural progenitors as well as post-mitotic neurons. Persistent expression from stably integrated lentiviruses also permits long-term analysis of protein function, including at various stages of cell differentiation. CGNs infected with lentiviral vectors continue to stably express transgenes for at least ten days in culture with no significant changes in their viability or properties (data not shown). Thus, events occurring at different phases in CGN development can be examined in a quantitative manner using initially mitotic or post-mitotic cultures. Lentiviruses harboring inducible promoters (Kafri et al., 2000) may prove particularly useful for expressing proteins at specific developmental stages. Further, results obtained in purified cell cultures can be extended to slice cultures, as shown here, as well as in vivo via injection into specific brain regions (Jakobsson et al., 2003). Lentiviruses also are suitable for altering protein expression and function in a variety of different neuronal cell types (Azzouz et al., 2002; Hottinger et al., 2000; Nadeau and Lester, 2002), and they transduce cortical neurons and CGNs with similar efficiency, as shown in the present study. Lentiviruses also offer important advantages as integrated promoter vectors over standard plasmid transfections, including (1) efficient vector transduction, (2) promoter expression and regulation in a more physiological chromatin environment, and (3) stable expression permitting promoter analysis during differentiation and other timedependent events. They also readily incorporate relatively large genomic promoter sequences. The mouse GABRA6luciferase transgene employed here is ∼12 kb in size, for

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example. Using co-infection of protein and promoter expression vectors, it is also possible to directly study functional interactions between transcription factors and their gene targets (Wang et al., 2004). Chromatin-dependent mechanisms are critical for gene regulation during cell differentiation (Muller and Leutz, 2001), and lentiviruses offer opportunities for defining their mechanisms in great detail in primary neurons that are not possible with episomal DNAs. An important finding here was the biphasic nature of lentiviral-mediated gene expression, with an early peak occurring during the initial period of virus exposure. Thus, lentiviruses can effect protein expression acutely, within 6 h in CGNs. This further enhances their versatility for studies requiring both short- as well as longer-term functional analyses. In particular, onset of transgene expression can be tightly controlled, which is of value when studying developmental and other temporal processes. While the mechanism for the early phase of viral-mediated expression is not certain, a likely explanation is the initial presence of functional but non-integrated viral DNA. Recent studies have demonstrated the ability of unintegrated lentiviral DNA to drive expression of transgenic mRNA and protein, particularly in non-dividing cells such as neurons (Saenz et al., 2004). This expression declines with time in culture following the removal of virus from the culture medium, presumably due to depletion of the pool of episomal transgene DNA as integration proceeds. Transduction of CGNs was dependent on time in culture, decreasing in efficiency between 1 and 3 DIV. This did not precisely correlate with cell proliferation, suggesting that other mechanisms were involved. One possibility is reduced virus uptake into cells. For example, GM3 ganglioside, which is involved in VSV-G-mediated uptake, declines in CGNs during their maturation (Prinetti et al., 2001). Reduced integration of viral DNA into the host cell genome also may occur during CGN differentiation, perhaps due to changes in chromatin organization. Whatever the mechanism(s) involved, lentiviral vectors remain useful for protein transduction and analysis of CGNs even at later time points. Lentiviruses have received great attention as stably expressing reagents for gene therapy within the nervous system and other tissues (Kordower et al., 2000). The present findings illustrate that these vectors also are powerful and highly versatile tools for exploring gene regulation of neuronal development in a quantitative manner.

Acknowledgments We would like to thank Mr. George Gagnon for his excellent assistance in various aspects of this work. These studies were supported by Public Health Service Grant RO1CA79999 to D.L.K. and Center Grant DK32520. The contents herein are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

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