In vitro analysis of promoter activity in Müller cells

Vector design

We modified a LV transfer vector (pFTMGW) [22] by adding restriction sites and enhancing the multiple cloning site (MCSv3); the new vector was named pFTM3GW (Figure 1). Briefly, we replaced the original multiple cloning site (MCS) in pFTMGW with MCSv3, in which the order of the 12 unique restriction sites was modified (5′-PacI-AsiSI-BlpI-RsrII-SwaI-AscI-HpaI-BsiWI-SdaI-NheI-BstEII-BamHI-3′) to facilitate double-digestion reactions (the BsiWI site was newly added). In addition, we flanked the transcription blocker (TB) with an additional BstBI restriction site, facilitating rapid removal of the TB before LV production. Finally, the hCMV/LTR hybrid promoter was modified such that it was flanked by SpeI endonuclease sites, allowing for efficient removal during preliminary candidate promoter evaluation, if desired.

Candidate sequence selection

Using online bioinformatics software (dcode) we identified and cloned full-length promoters (~1500 bp), proximal promoter regions (500 bp), and the most proximal ECRs, relative to the transcription start site (TSS), for each of nine Müller cell expressed genes: CAR2, CD44, GFAP, GLUL, PDGFRA, RLBP1, S100B, SLC1A3, and VIM. ECR sequences were identified in canonical promoter regions mandating a minimum of 70% sequence homology between human and mouse genomes over a 100 bp “window.” Based on the observation that several genes have sizable 5′ untranslated regions (UTRs) containing numerous transcription factor binding sites, in addition to the fact that 5′ UTRs afforded improved expression in first-generation constructs, fragments from each of the nine genes (1500 bp, 500 bp, and ECR) were cloned with and without their associated 5′ UTR, or part thereof (see Appendix 1). All transcription start sites were identified using RefSeq sequences (NCBI) compiled into single data files available at (ECRbase). The current mouse (mm8), rat (rn4), and human (hg18) assemblies of their respective genomes were used for all sequence comparisons, alignments, and primer design.

Sequence analysis and primer design

DNA sequences identified using the ECR Browser were imported into Vector NTI (VNTI; Invitrogen, Carlsbad, CA). Primers were selected using VNTI primer design software, and one or two restriction sites were manually added to the 5′ ends of each primer to confer additional flexibility in the cloning process and to allow constructs to be cloned into different regions of the MCS in the pFTM3GW vector (see Appendix 1 and Appendix 2). Primers from Operon Technologies (Huntsville, AL) were resuspended at 100 µM in 10 mM Tris (pH 8.0) and 1 mM EDTA.

Polymerase chain reaction amplification and cloning

Sequences were PCR-amplified from C57BL/6J mouse or Sprague-Dawley rat gDNA using 0.5 U per reaction Platinum Taq or Platinum Taq High-Fidelity Polymerases (Invitrogen). Standard reaction conditions were 2.5 mM Mg²⁺, 0.2 µM each primer, 0.2 mM deoxyribonucleotide triphosphates (dNTPs), and 10–50 ng gDNA in a 20 µl reaction volume. A list of cloned sequences, including their primer sequences and their respective chromosomal locations, are shown in Appendix 1 and Appendix 2. PCR products were typically T/A cloned into the pGemT-Easy vector (Promega, Madison, WI) and transformed into DH5alpha chemically competent cells (Invitrogen). Plasmid DNA was isolated using the QIAprep Miniprep Kit (Qiagen Corp., Valencia, CA). Each promoter fragment was digested and gel purified from pGem-T-easy, and subsequently subcloned into linearized pFTM3GW. For verification, all constructs were PCR screened, endonuclease digested, and sequenced. While most promoter sequences matched published database sequences exactly, some sequences had individual point mutations as well as small (1 or 2 bp) insertions or deletions, particularly in genomic regions containing a minisatellite repeat(s).

Cell culture and transfection

Cell culture was performed as previously described [22] for both rat (wMCs) and human (MIO-M1) Müller cells [36]. MIO-M1 cells were a gift from University College London. NIH/3T3 (ATCC#: CRL-1658) and Neuro-2a (ATCC#: CCL-131) were obtained from the University of California, Berkeley core research facility. Briefly, approximately 200,000 Müller cells or 400,000 NIH/3T3 or Neuro-2a cells were plated per well on 12 well plates. Cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) including high glucose (Invitrogen) with 10% fetal bovine serum (FBS; HyClone; Thermo Fisher Scientific, Waltham, MA) and 4 mM L-glutamine without antibiotics until they reached 90% confluence (~24 h). A transfection complex with 0.75 µg plasmid DNA (experimental or control) and 3.75 µl Lipofectamine 2000 (Invitrogen) in 200 µl of OptiMEM (Gibco; Invitrogen) medium was prepared according to the manufacturer’s specifications. Growth medium was removed and the transfection complex was added into each individual well. After 10 min, 1 ml of fresh media without antibiotics was added. Cells were cultured for approximately 40 h before flow cytometry analysis.

Flow cytometry and data analysis

Methods for flow cytometry and fluorescence microscopy were described previously [22]. Briefly, transfected wMC and MIO-M1 Müller cells were trypsinized for ~3–5 min, resuspended in 500 µl DMEM media, and maintained on wet ice. At least 20,000 cell counts were collected for each sample, and three independent samples were counted for each construct. Mean fluorescence intensity (MFI) and percent eGFP positive cells were calculated and collected for each sample under identical gating conditions within each experiment. All data were normalized to the promoter-less parent vector (pFTMGW or pFTM3GW) or pFUGW, and were subsequently plotted as relative fold-change in terms of number of eGFP labeled cells and MFIs. pFTM3GW and pFTMGW [22] employ a TB element to minimize (by 85%–90%) transgene expression by an upstream hybrid CMV/5′LTR promoter, which is necessary for virus production. The fortuitous “leakiness” of the TB element has two benefits: 1) it allows for efficient and accurate normalization across experiments; and 2) it allows for the identification of MCS-cloned elements that negatively influence transgene expression. We used Microsoft Excel for all statistical analyses, using the Student's t-test for all comparisons (two-sample assuming equal variances). Analyses were performed on raw data counts and collected as percent positive cells (fluorescent, flow cytometry counted). Data and errors were normalized to the promoter-less parent vectors (pFTMGW or pFTM3GW) or pFUGW. Data obtained from control plasmid transfections were set to a value of 1.0 (or 100%), which allowed for relative fold-change (increase or decrease) calculations for experimental constructs, as well as a baseline for comparison between experiments. All error bars represent (±) 1 standard deviation.

Microscopy

For microscopic examination of eGFP expression, cells were grown on cover glass under identical conditions, and were processed as previously described [22]. Briefly, cells were transfected for approximately 24 h, rinsed with phosphate buffered saline (PBS; pH 7.4), fixed with 10% neutral buffered formalin for 15 min, rinsed again with PBS, and inverted on a microscope slide containing a drop of Vectashield (Vector Laboratories, Inc., Burlingame, CA) containing DAPI (4',6-diamidino-2-phenylindole) as a nuclear counterstain (blue). Images were collected using Zeiss Axiovision 4.4 software (Carl Zeiss, Oberkochen, Germany) with a fixed exposure time, and all images were identically post-processed with Adobe Photoshop (Adobe Systems, San Jose, CA).

Control plasmids and normalization

The pFTM3GW LV transfer vector (Figure 1) is an improved version of pFTMGW [22] (see Methods for details). This vector utilizes a TB element to greatly reduce expression (by 85%–90%) from the hybrid hCMV/5′LTR (a promoter element required for in vitro virus production). In each of the experiments presented here, we utilize the low basal level of “read-through” expression (~10%–15%) as our baseline for normalization, except for the comparative expression in NIH/3T3 and Neuro-2a cells, for which the pFUGW plasmid (containing a portion of the human ubiquitin-C promoter) was used for normalization. Thus, the percentage of eGFP positive cells counts for pFTMGW or pFTM3GW control plasmids were assigned a value of 1.0, and all measurements of experimental constructs are shown in terms of fold change (in number of eGFP positive cells) relative to pFTMGW, pFTM3GW, or pFUGW. In addition, all MFI's were similarly normalized: these data are indicated in each graph immediately under each bar (shaded with orange). Lastly, the “leakiness” of the TB permits the identification of putative repressive regulatory elements.

Müller cell-associated promoter testing

We systematically examined the activity of 61 DNA sequences in the promoter regions of 9 Müller cell-associated genes (Figure 2, Appendix 1). Fragments cloned from each gene are schematically represented in Figure 2A, and numbered relative to the TSS. We cloned the 500 bp and the 1500 bp sequences, both with and without the adjacent 5′ UTR, for each of the nine genes (Figure 2B-2J). In addition, ECRs were cloned with and without the 5′ UTR. In some cases (CD44, GFAP, RLBP1, and VIM; see Appendix 1), the most proximal ECR was not immediately adjacent to the TSS: when cloning these ECR+UTR fragments, additional intervening sequences possessing less than our 70% homology threshold between the ECRs and 5′ UTRs, including the TSSs, were included. Finally, individual UTR sequences for seven of the nine genes were tested alone. The UTRs for CAR2 (28 bp) and GFAP (14 bp) were quite small, and not individually examined.

Variable influences of the 5′ untranslated regions

In general, the UTRs alone were capable of increasing the number of eGFP positive cells two- to sevenfold (Figure 2). RLBP1 (Figure 2G) showed very low expression unless the UTR was combined with other regulatory elements (500 bp, 1500 bp, or ECR). Combining RLBP elements (500+UTR, 1500+UTR, or ECR+UTR) resulted in greater than four- to sevenfold more Müller cells expressing eGFP, with a concomitant increase in MFIs (up to 3.6-fold higher; 500+UTR). Similarly for the S100B gene (Figure 2H), including the UTR led to increased expression over individual constructs. For S100B, the combined constructs (500+UTR and ECR+UTR) appeared to have more of an “additive” effect on eGFP expression, whereas a “multiplicative” influence was apparent for RLBP1. The UTRs of CD44 (Figure 2С) and PDGFRA (Figure 2F) drove significant levels of eGFP expression on their own, whereas the individual 5′ elements (500 bp, 1500 bp, and ECR) resulted in considerably lower eGFP expression.

In contrast, some constructs without UTRs drove significant levels of eGFP in cultured Müller cells. For example, the 500 bp, 1500 bp, and ECR elements in CAR2 (Figure 2B), GFAP (Figure 2D; except ECR), GLUL (Figure 2E), and VIM (Figure 2J) resulted in three- to eightfold changes in the number of cells expressing eGFP. Note that the 118 bp GLUL UTR led to a nominal (approximately twofold) change in reporter gene expression by itself, whereas the individual constructs (with or without the UTR) resulted in six- to eightfold increases. Thus, in the case of GLUL, the UTR appeared to exhibit only a minor influence on gene expression, whereas upstream elements contributed significantly. Constructs isolated from the SLC1A3 gene exhibited variable influences on eGFP expression (Figure 2I). In contrast to other genes, the SLC1A3 1500+UTR and 500+UTR sequences led to reduced expression relative to the 500 bp and 1500 bp fragments without the UTR (Figure 2I), suggesting a complex cis-regulatory network involving both positive and negative influences at the SLC1A3 promoter. Even though the cellular specificity of these regulatory elements has not been verified, it is worth noting that the short, 205 bp GFAP ECR+UTR promoter fragment drove eGFP expression as well as any other GFAP construct tested (Figure 2D).

Microscopic examination of 500 bp elements

We screened promoter constructs using fluorescence microscopy before flow cytometry analysis, and qualitatively assessed their eGFP expression 24–40 h following transfection. Figure 3 shows fluorescent micrographs of rat Müller cells grown on cover glass and transfected with the 500 bp and 500+UTR constructs for each of the nine genes. Cultures were treated identically to those used for flow cytometry experiments, and qualitatively support the quantitative flow cytometry data shown in Figure 2. The presence of the UTR enhanced the number of eGFP-labeled cells in several experimental constructs: CD44 (Figure 3B); PDGFRA (Figure 3E); RLBP1 (Figure 3F); S100B (Figure 3G); and VIM (Figure 3I). In contrast, little or no effect was observed for CAR2 (Figure 3A), GFAP (Figure 3C), or GLUL (Figure 3D). Lastly, the presence of the UTR in the SLC1A3 construct (500+UTR; Figure 3H) resulted in fewer eGFP labeled cells (also see Figure 2I). Control transfections (Figure 3J) appropriately exhibited a few dim (pFTM3GW) or numerous bright (pFUGW; containing the human ubiquitin-C promoter) fluorescent wMC Müller cells.

Comparison between rat and human Müller cells

To assess orthologous promoter activity, we applied the same 500 bp and 500+UTR promoter constructs to a spontaneously generated human Müller cell line (MIO-M1 [36]). Figure 4 indicates fold changes in eGFP positive cells in both rat (red) and human (blue) cultured Müller cells. All data were normalized to the promoter-less parent plasmid, pFTM3GW (not shown). As one might expect from divergent DNA promoter sequences, the overall numbers (and MFIs) of eGFP positive human Müller cells were reduced relative to the rat (wMC) Müller cells. Importantly, though, the trends in the data were notably conserved for several genes, suggesting that some orthologous, sequence-related mechanisms are likely participating in the reporter gene expression. Some genes exhibit similar expression profiles (RLBP1, S100B, SLC1A3), while others exhibit definitive differences (CAR2 and GFAP).

Further analysis of the mouse and rat CD44 and vimentin promoters

We computationally identified, cloned, and screened additional mouse and rat ECR sequences from the CD44 and VIM genes. A total of 32 ECRs (see Appendix 2) located both upstream and downstream (typically more distal conserved regions) of the mouse and rat CD44 and VIM coding sequences were cloned and studied by either flow cytometry or fluorescence microscopy or both. Approximately 60% (20/32) of the individual constructs resulted in expression levels that were indistinguishable from pFTMGW vector alone (see Appendix 2; constructs with “nd” in the eGFP and MFI columns). After microscopic screening (data not shown), the remaining ECRs (12/32) were analyzed by flow cytometry (Figure 5; Appendix 2). Notably, some ECRs representing the same conserved regions in mouse and rat drove eGFP expression at similar expression levels (Figure 5; CD44M2/CD44R2 and VIMM2/VIMR2). Both pairs of promoter constructs resulted in roughly two- to threefold increases in number of eGFP expressing cells. Interestingly, the two rodent VIM genes have different TSSs; VIMR2 (rat) is a 409 bp fragment, containing 330 bp of the VIM promoter and 79 bp of the VIM 5′ UTR, whereas VIMM2 (mouse) is entirely within the VIM 5′ UTR. It is also important to notice the reduction in the number of eGFP positive cells (and MFI) by the VIMR6 and CD44R4 constructs. In particular, the VIMR6 fragment negatively influenced eGFP expression by both flow cytometry (Figure 5) and fluorescence microscopy (data not shown). Moreover, when added to the VIMR2 promoter fragment, VIMR6 reduced the number of wMC Müller cells by ~33% (Figure 6).

Complex and chimeric promoters are readily testable using pFTMGW or pFTM3GW

We examined the feasibility of cloning and testing in silico-designed, conjoined promoter constructs (Figure 6). We added secondary ECRs to two promising plasmid constructs containing VIMR2 or CD44R2 (both of rat origin). At least for VIMR2 and CD44R2, the inclusion of additional ECR elements upstream of existing elements (having already been cloned into the MCS) resulted in a reduction in the number of eGFP expressing cells. The chimeric construct, CD44R2 and VIMR2 (red-blue bar), was not significantly different from either individual construct with respect to number of eGFP positive cells or MFIs. Duplicating the VIMR2 element slightly decreased the number of cells, but did not affect the level of expression (MFI) when compared to the single VIMR2 construct. Directional selectivity/specificity of the VIMR2 promoter was confirmed by testing a construct in which VIMR2 was cloned in the opposite orientation (VIMR2-Reverse; 3′-5′ orientation). The VIMR2-Reverse construct resulted in slightly fewer eGFP positive cells (p=.033) when compared to the vector alone (pFTMGW).

We also identified a short fragment from the rat vimentin gene that appeared to negatively modulate transgene expression when tested individually and when included in combination with other putative regulatory elements. The VIMR6 sequence lies ~2.8 kb upstream (5′) of the gene’s TSS, and its specific function remains to be determined. Shown in Figure 5, the 360 bp VIMR6 ECR fragment decreased the amount of pFTMGW baseline expression by 45%, suggesting that VIMR6 represents a putative inhibitory cis-regulatory element. In Figure 6, we show that combining VIMR6 with VIMR2 decreased the number of eGFP positive Müller cells by nearly 30% (compared to VIMR2-only levels), adding further evidence that VIMR6 has the capacity to mitigate gene expression. However, note that VIMR6 did not decrease MFI levels. For the CD44 gene, the CD44R4 construct exhibited similar negative-modulating characteristics as VIMR6.

Comparison of expression in two non-glial cell types

Six experimental constructs (GLUL 500±UTR, RLBP1 500±UTR, and S100b 500±UTR) were tested by transfection in 3 cell types: wMC Muller cells, NIH/3T3 fibroblasts, and Neuro-2a neuroblastoma cells (Figure 7). Both cell count and MFI values for all constructs were normalized to pFUGW expression levels. Under these culture conditions it is clear that the promoter constructs tested do not restrict expression to “glial” cells. After normalization to the pFUGW construct, comparable percentages of cells (Figure 7A) and MFIs (Figure 7B) were observed for wMC, NIH/3T3, and Neuro-2a cells. Overall eGFP positive cell counts indicated that Neuro-2a cells were significantly more receptive to transfection, approaching 95% transfection efficiency (compared to ~20% for both wMC and NIH/3T3 cells, using an identical transfection protocol; data not shown). Most notably, a distinct difference was observed when NIH/3T3 cells were transfected with the RLBP1 500+UTR construct, which resulted in over twice as many cells becoming eGFP positive, relative to the pFUGW control (Figure 7A). In addition, it is noteworthy to point out that overall MFI levels varied significantly for the three cell types: the average eGFP intensities (arbitrary units) for the pFUGW constructs were 53.7, 9.7, and 86.5 for wMC, NIH/3T3, and Neuro-2a cells, respectively. Thus, overall strength of transgene expression appeared to be determined by the combination of promoter activity and constitutive (undetermined) cellular qualities. Nevertheless, when normalized to pFUGW, the trend with the experimental constructs was that wMC cells expressed the eGFP reporter molecule at intermediate levels, generally between that of NIH/3T3 and Neuro-2a cells.