Department of Medicine, Medical University of South Carolina and the Medical and Research Services, Ralph H. Johnson Veterans Affairs Medical Center, Charleston, South Carolina 29425
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ABSTRACT |
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Early passage
mesangial cells, like many other nonimmortalized cultured cells, can be
difficult to transfect. We devised a simple method to improve the
efficiency of transient protein expression under the transcriptional
control of promoters in conventional plasmid vectors in rat mesangial
cells. We used a vector encoding modified green fluorescent protein
(GFP) and sterile fluorescence-activated cell sorting (FACS) to select
a population consisting of >90% GFP-expressing cells from passaged
nonimmortalized cultures transfected at much lower efficiency. Only
10% transfection efficiency was noted with a -galactosidase
expression vector alone, but cotransfection with GFP followed by FACS
and replating of GFP+ cells
yielded greater than fivefold enrichment of cells with detectable
-galactosidase activity. To demonstrate the expression of a properly
oriented and processed membrane protein, we cotransfected GFP with a
natriuretic peptide clearance receptor (NPR-C) expression vector.
Plasmid-dependent cell surface NPR-C density was enhanced by 89% after
FACS, though expression remained lower in selected mesangial cells than
in the CHO cell line transfected with the same vector. We conclude that
cotransfection of rat mesangial cells with GFP, followed by FACS,
results in improvement in transient transfection efficiencies to levels
that should suffice for many applications.
automated cell sorting; adenovirus; lipofection; liposome
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INTRODUCTION |
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TRANSFECTION OF CELLS with cDNA encoding a protein not normally expressed in the host cell is a uniquely powerful method to assess the functions of novel or mutant proteins. Transfection can either be transient (expressed for a single or a few passages) or stable (expressed for multiple passages or with long-lasting effects). Transfections can also use chromatin-based incorporation of the DNA into the host genome or episomal non-chromatin-based expression. Unfortunately, the generation of stably transfected primary cultures of mammalian cells is time consuming and requires that the expressed protein not adversely affect the growth of the cells. Successful application of this process results in cells that uniformly express the target protein, but are many passages removed from the animal, increasing the probability of phenotypic changes that could reduce the physiological relevance of the cell model.
For many applications, a transient transfection approach would be preferable. This approach has been highly productive in signal transduction research in certain transformed cell lines, but it is more difficult to apply in primary or early passage nonimmortalized cultured cells, because transfection efficiency is often unacceptably low in these cells. Low transfection efficiency can affect the proportion of cells that express detectable levels of the transfected protein, and/or the number of copies of the DNA expressed per cell. There are many potential mechanisms that can reduce transfection efficiency. These include poor uptake of the vector, poor processing or expression of the vector once taken up by the cell, and inactivation or destruction of the vector by cellular enzymes.
Many methods have been used to attempt to circumvent low transfection efficiency in differentiated cells when classic approaches, such as calcium-phosphate precipitation (12), DEAE-dextran (29), or polylysine (31) methods, are inadequate. For example, viral vectors (e.g., Sindbis virus, retrovirus, or adenovirus) bearing nucleic acid sequences encoding the protein(s) of interest can be used to productively infect cells. In some cases, facilitators of viral uptake, such as fibronectin fragments, can further increase the efficiency of cellular expression (21). In cases where specific uptake mechanisms exist, inactivated viruses or viral coat proteins have also been used to facilitate the uptake of classic DNA vectors into certain cells in vivo or in vitro; often, the vector and/or viral particles are incorporated into liposomes (1, 18). A third method that has proven useful in some situations is to disrupt the plasma membrane by electroporation (13, 22), or with a particle gun, to introduce cDNA into the cells (14, 20). A fourth possibility would be to produce transgenic or gene-targeted animals as the source for primary cell cultures. A fifth method uses microinjection of vectors into cell cytoplasm or the nucleus (7). Although each of those techniques has considerable potential merit, they all suffer from drawbacks that limit their practical use or widespread availability. For example, the generation, characterization, and maintenance of viral stocks for expression constructs or liposomes is labor intensive and may present biosafety containment issues, resulting in requirements in time and resources beyond the reach of many laboratories. Similarly, the generation of stable strains of transgenic mice requires significant time and specialized technical expertise, and the transgenes may or may not be expressed in the tissue of interest. Thus these methods could be highly satisfactory for the intense study of a small number of targeted manipulations, but are less suitable for broad mapping or screening efforts.
We sought to devise an efficient and less resource-intensive method for induction of transient protein expression for use in signal transduction studies in renal mesangial cells, a limited-passage nonimmortalized culture system that is notoriously resistant to transfection. For our studies, we used a cotransfected marker to identify and study only the transfected target cells. This general approach has been useful in other cell lines (4) when increased transfection efficiency was necessary. The advent of green fluorescent protein (GFP) as a biomarker offered the opportunity to simplify the selection of transfected cells. GFP is derived from the jellyfish Aequorea victoria, to which it confers luminescence. Laser-stimulated GFP fluorescence can be detected in intact cells in real time without the need for cofactors, substrates, or other proteins (16). We took advantage of this property to isolate transfected mesangial cells post hoc using sterile fluorescence-activated sterile cell sorting (FACS). We found that the cells thus obtained could be replated in either flasks or six-well clusters, and subsequently maintained for at least 72 h. In cells selected for high GFP expression, we report that the transfection efficiency and/or expression levels of two different cotransfected cDNAs were considerably improved over levels seen in nonselected cells.
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MATERIALS AND METHODS |
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Chemicals. Lipofectin, Lipofectamine, cell culture media, serum, and antibiotics were from Life Technologies (Gaithersburg, MD), and culture flasks were from Costar (Cambridge, MA). 125I-labeled atrial natriuretic peptide (ANP) was purchased from DuPont New England Nuclear (Boston, MA). DOSPER [1,3-dioleoyloxy-2-(6-carboxyspermyl)-propylamid] was from Boehringer Mannheim (Indianapolis, IN). Chemical reagents were purchased from Sigma (St. Louis, MO) unless otherwise specified.
Plasmids.
The vector encoding a GFP mutant (phGFP-S165T) was purchased from
Clontech (Palo Alto, CA).1 This vector encodes
a mutant GFP that contains >190 silent nucleotide changes to optimize
the coding sequence based on human codon-usage preferences (15), and a
mutation at residue 165 (SerThr), which results in enhanced
fluorescence and a single excitation peak at 490 nm. Native GFP is a
238-amino acid protein (26) with a major absorbance peak at 395 nm
(ultraviolet) and a smaller peak at 475 nm (blue light) (6). Because
living cells tolerate blue light better than ultraviolet light, the
minor peak is better suited for intact cell applications. The
red-shifted excitation peak of the modified GFP reduces photobleaching
and allows for the use of the 488-nm line on the argon lasers used in
many fluorescence microscopes and fluorescence activated cell sorting
machines. The pSV-
-gal expression vector (Promega, Madison, WI) is a
vector in which
-galactosidase expression is under the
transcriptional control of the SV40 early promoter and enhancer. The
natriuretic peptide clearance receptor (NPR-C) expression vector
pCDNA3-NPR-C was constructed in our laboratory. Human NPR-C cDNA (25)
(a kind gift from J. Gordon Porter, PhD) was subcloned into the
EcoR I site of the expression vector
pCDNA3.1(+) (Invitrogen, Carlsbad, CA), which contains a
cytomegalovirus (CMV) promoter. We subjected the resulting
plasmid to sequence analysis to determine that the insert was in the
correct orientation. The vector was also characterized by its direct
expression in the Chinese hamster ovary (CHO) cell line (American Type
Culture Collection, Rockville, MD; see
RESULTS).
Isolation and culture of rat mesangial cells. We obtained mesangial cells from Sprague-Dawley rats (150-200 g) using standard sieving techniques, as previously described in detail (23, 24). Cells were incubated at 37°C in a humidified atmosphere of 95% air-5% CO2, and subcultured every 1-2 wk by trypsinization. Cells were plated at a density of 2-5 × 104 cells/ml in RPMI medium (pH 7.3) supplemented with 1 mM HEPES, 5 mg/dl insulin, and 20% FCS. Cells used were from passages 4-15.
Transfection.
Nearly confluent cells were exposed to 1 ml of serum- and
antibiotic-free RPMI medium containing 10 µl Lipofectamine,
Lipofectin, or DOSPER, and 1 µg phGFP-S165T/100
mm2 cell culture surface
area. In cotransfection experiments, 1 µg of pSV--gal
or pCDNA3-NPR-C/ml transfection mix was also included. The ratio of 10 µl of lipid per 1 µg of DNA was chosen because this ratio delivers
the most efficient expression of plasmid DNA in CHO cells (10), which
are commonly used for transient expression studies. After incubation at
37°C for 2 h, the transfection medium was aspirated and replaced
with growth medium. Cells to be examined by fluorescence microscopy
were incubated for 24-48 h. For cell sorting, cell monolayers were
washed with PBS containing 0.5% BSA, and then detached from dishes by trypsinization.
Flow cytometry and sterile cell
sorting.
Cells were pelleted by centrifugation, resuspended in PBS containing
0.5% BSA to a final density of ~2.5 × 106 cells/ml, and filtered through
a nylon membrane to remove cell aggregates. Flow cytometry and cell
sorting for GFP and propidium iodide (PI) fluorescence were performed
using a FACStar Plus (Becton Dickinson, San Jose, CA) with INNOVA 70-4 argon laser tuned to 488 nm. Data acquisition and analysis were
performed with CellQuest software. A minimum of 10,000 events was
collected for each analysis. GFP signals were detected with a 530/30-nm
bandpass filter, and PI signals with a 630/22-nm bandpass filter. To
facilitate the recovery of large numbers of transfected cells, we
performed presorting triggered by fluorescence; we performed the final
sort into GFP+ and
GFP populations using
forward scatter as a triggering signal. Where applicable,
PI+ cells were excluded on both
sorting runs. The resulting cells were resuspended in growth
medium and allowed to reattach to culture dishes. Expression of GFP
and/or cotransfected proteins was analyzed 18-72 h later.
125I-ANP binding
assay.
Binding of 125I-ANP was used to
measure NPR-C expression. Binding assay was performed at 4°C for 2 h, as described (23). Briefly, CHO or mesangial cells in 24-well plates
were chilled to 4°C for 15 min and washed with Hanks' balanced
salt solution (HBSS), pH 7.4. They were then incubated in 250 µl HBSS
containing 10 mM HEPES, 90 mg/l phenylmethylsulfonyl fluoride, 0.2%
BSA, and 100 mg/l bacitracin, pH 7.4. The incubation mixture included
~105 cpm/well
125I-ANP (Amersham, Arlington
Heights, IL). At the end of the incubation, the binding mixture was
aspirated, and cells were washed with ice-cold PBS and solubilized in
0.5 ml/well of 1 N NaOH for -counting. Specific binding was
determined as the difference between bound counts in the absence and
presence of 100 nM des-(18-22)-ANP-(4-23). NPR-C is normally
expressed in mesangial cells, but its expression is minimized in the
presence of 20% fetal bovine serum in the growth medium (23). The
mechanisms underlying this regulation have been published previously
(23, 24), but for our purposes, incubation with 20% calf serum served
to reduce the expression of endogenous NPR-C, such that transfected
NPR-C could more easily be measured. The density of heterologous NPR-C
arising from the transfected cDNA was determined by subtraction of the
background of specific binding from nontransfected cells. NPR-C
expression was quantitated as counts specifically bound per
106 cells.
In situ staining for
-galactosidase expression.
-Galactosidase staining was carried out according to the procedure
described by Sanes et al. (27), with minor modifications. Cells on
micro coverslips were rinsed with PBS and then fixed for 15 min at
4° C in 0.5% glutaraldehyde in PBS containing 2 mM
MgCl2. The cells were then washed
with PBS and overlaid with 5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside
(X-gal) staining solution overnight at room temperature. The X-gal
staining solution was composed of 0.5 mg/ml X-gal, 5 mM potassium
ferricyanide, 5 mM potassium ferrocyanide, and 2 mM
MgCl2 in PBS.
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RESULTS |
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In pilot studies, we determined the optimal concentrations of transfection vehicles and the length of incubation required for the best expression of GFP. Nearly confluent cells seeded in six-well dishes were exposed to three different lipid transfection vehicles (Lipofectin, DOSPER, and Lipofectamine) in varying amounts for varying time periods, with and without serum in the transfection mix, along with 1 µg phGFP-S165T expression plasmid. Cells were examined at 24 and 48 h, both for expression of GFP (by fluorescence microscopy) and for gross evidence of toxicity. Only 7 ± 2% of cells were GFP+ with Lipofectin (n = 5). Better expression levels were obtained with DOSPER (27 ± 3%, n = 3) and Lipofectamine (31 ± 5%, n = 5). DOSPER required incubation periods of 8-12 h with mesangial cells, whereas optimal results were obtained with Lipofectamine with only a 2-h incubation in serum-free medium. Longer incubations with Lipofectamine resulted in obvious toxicity (i.e., cell detachment and formation of blebs). DOSPER appeared to be less toxic to mesangial cells. However, we chose Lipofectamine for further study due to the convenience of the brief incubation time.
All of the results reported below were obtained from cells transfected
with phGFP-S165T using a 2-h incubation with Lipofectamine. After 36
h, the percentage of GFP+ cells
was measured. In nine separate experiments, transfection efficiency
varied between 10% and 40%, depending, to some extent, on the minimum
fluorescence intensity accepted for GFP positivity. These efficiencies
are higher than are typically achieved, most likely due to the
sensitivity of the GFP assay, and due to our efforts to optimize the
transfection conditions. Figure 1 depicts the results of an experiment in which we selected cells for both GFP
expression and PI exclusion. Cells that were unable to exclude PI from
the nucleus were assumed to be nonviable.
GFP+-PI
cells were then allowed to reattach overnight. The next day, the cells
were again harvested for flow cytometry. On this second analysis, the
cells were >90% GFP+ and <1%
PI+. These results established
that a viable cell population that almost universally expresses the
marker protein could be readily obtained and maintained overnight,
although expression levels varied considerably from cell to cell.
Figure 2 demonstrates the findings on fluorescence
microscopic examination of this cell population.
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To determine whether selection of
GFP+ cells could lead to the
enrichment of mesangial cells expressing a separate target gene, we
carried out cotransfection experiments. We first transfected the
pSV--gal expression vector together with phGFP-S165T. Transfected cells were separated into two different groups based on the intensity of the fluorescent GFP signal. In the experiment shown in Fig. 3, a single round of cell sorting was performed to yield
two groups of cells: cells that did not express or had low levels of
fluorescence (GFP
) and
cells that showed relatively high fluorescence intensity (GFP+). Sorted cells and
controls were plated in equal numbers as follows: 1) mock-transfected cells,
2) cotransfected but unsorted cells, 3)
GFP
cells, and
4)
GFP+ cells. For this experiment,
PI exclusion was not assessed because it was assumed that nonviable
cells could not be replated and, therefore, would not contribute to
results obtained after further cell culture. The cells were then
stained for
-galactosidase expression 15 h later. Mock-transfected
cells had no
-galactosidase staining, and
GFP
cells also showed
virtually no
-galactosidase staining. A small portion (
10%) of
the cotransfected, nonsorted population showed staining for
-galactosidase. In contrast, >50% of cells in the GFP+ group stained positively for
-galactosidase, indicating a reasonably good correlation between GFP
expression and
-galactosidase expression. Therefore, by selecting
cells that expressed high levels of the GFP marker, a greater than
fivefold enrichment of target gene (
-galactosidase) expressing cells
was achieved.
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-Galactosidase is a widely used marker for transfection efficiency.
It is a small and robust protein whose enzymatic activity can
be easily and sensitively detected against the background activity in
many mammalian cells. Measurement of the expression of other marker
genes could potentially suffer from a lower signal-to-noise ratio. The
maintenance of activity of more complex proteins that require extensive
posttranslational modifications can be another significant problem. To
further establish the usefulness of our approach, we cotransfected
cells with GFP and NPR-C. NPR-C was deemed suitable because it is a
plasma membrane protein normally expressed by mesangial cells, which
requires proper proteolytic cleavage, plasma membrane targeting,
orientation, and N-linked glycosylation to bind its ligand. Therefore,
ligand binding of transfected NPR-C represents a stringent test of the
capability of mesangial cells to process an endogenous protein normally
after exposure to the rigors of GFP cotransfection, cell sorting, and replating.
The experimental design, including grouping of
the cells, was similar to the experiment shown in Fig. 3; selected
cells were above the 90th percentile of GFP fluorescence intensity. The
cell surface expression of NPR-C was measured by
125I-ANP binding, displaceable by
excess des(18-22)-cANP-(4-23), a specific NPR-C ligand.
Mesangial cells can also express another 125I-ANP binding site, NPR-A, but
ANP is not displaced from this site by the competing ligand (23), and
binding to this site would therefore be included in "nonspecific"
binding in this assay. The endogenous NPR-C binding (1,800 cpm/106 cells) was relatively low
in these mesangial cells under our culture conditions, which were
intentionally designed to suppress expression of endogenous NPR-C (23,
24). The background binding was subtracted to yield the specific NPR-C
binding, which resulted from expression of the transfected cDNA, or
"heterologous" binding. As shown in Fig.
4, the level of heterologous
125I-ANP binding was 2,393 ± 40.4 cpm/106 cells in transfected
cells before cell sorting. After sorting, the
GFP+ cells had 4,533 ± 1,080 cpm of
125I-ANP/106
cells, an 89% enrichment. In contrast, the level of heterologous 125I-ANP binding in the
GFP group was 853 ± 423 cpm/106 cells. Therefore, the
upper 10% of cells, in terms of GFP expression, had more than
fivefold higher NPR-C expression than the remainder of the cells, and
accounted for ~60% of the total heterologous NPR-C expression in the
original unsorted transfected population. To generate a standard of
comparison for NPR-C expression, the construct was also transiently
transfected into CHO cells. This fibroblast cell line is frequently
used for transient transfections because of a well-recognized capacity
to efficiently take up and express foreign DNA. Specific binding in
this cell line was negligible in mock-transfected cells, and was 13,450 ± 245 cpm/106 cells in cells
transfected with the NPR-C construct. Therefore, plasmid-dependent
NPR-C density in nonsorted, transfected mesangial cells was only
~18% of the presumably optimal level observed in CHO cells, but this
fraction increased to 33% after cell selection.
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DISCUSSION |
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The efficient transfer of genetic material into kidney cells has been a challenging proposition (17). Many potential problems have limited the use of nearly every transfection method in kidney cells. Although the technical nature of this communication does not allow for an in-depth discussion of each method, we will briefly enumerate some of the more commonly encountered problems with nonviral and viral vector methods.
Electrostatic liposomes are made by attaching small cationic lipids to negatively charged DNA vectors (9), and those complexes are taken up by the cell, mainly by phagocytosis. Some problems with this approach are that liposomes may not be efficiently phagocytosed by target cells or may induce cellular toxicity. Electrostatic liposomes may also have some packaging limitations as far as the allowable vector size. Another potential disadvantage is that the DNA introduced by the electrostatic liposome method is subject to processing by cellular endosomal and lysosomal systems. In contrast, the internal liposome method uses viral coat proteins to achieve fusions with the cell membrane, allowing uptake of the packaged nucleic acids into the cytosol without the requirement for phagocytosis. One common internal liposome method uses the HVJ (hemagglutinating virus of Japan) paramyxovirus envelope, which contains HN and F glycoproteins that affect fusion. This method has many potential advantages, including highly efficient transient expression of large DNA packages, but has several disadvantages, including extremely complex preparation requirements and lower vector stability than other methods. More classic transfection methods (calcium-phosphate or DEAE-dextran) can also be toxic to cells, or may result in expression of low copy numbers. In addition, nearly all types of nonreplicating vectors are subject to degradation by nucleases, and can be partitioned into non-nuclear compartments (5). Furthermore, because there is no vertical transfer of the packaged DNA, the number of extrachromosomal copies of DNA can fall dramatically by dilution in replicating cells (2).
In general, viral vectors usually have a higher efficiency than nonviral methods. However, because many separate steps are typically required for the expression of the packaged nucleic acid construct, viral transfection or infection strategies can be limited in several regards. The retroviruses serve as excellent examples of the complexities involved in this process. Retroviruses are RNA viruses that bind to specific cellular receptors that are required for viral docking and subsequent transfer of the viral RNA into the cytoplasm of the recipient cells. The RNA is then converted into proviral DNA by reverse transcription. The proviral DNA must then be transported into the nucleus, where it is converted into double-stranded DNA, which must then randomly incorporate into the genome of the host cell. Therefore, difficulties could arise at multiple steps in this process, including 1) the receptor-binding step, 2) the cytoplasmic-delivery step, 3) the reverse-transcription step, 4) the nuclear-transport step, 5) the step in which single-stranded DNA is converted into double-stranded DNA, or 6) integration into the host cell genome. Thus it is not surprising that retroviruses usually yield low copy numbers, with only one or two integrated copies per cell (11). Moreover, retroviruses can only be expressed in replicating cells.
Other viruses, such as the adenovirus or adeno-associated virus, can function in both replicating and nonreplicating cells. Adenoviruses are very useful in that large DNA fragments can be efficiently delivered to the host cell at high titres. However, because the typical adenoviral vectors are not integrated into the host genome and do not replicate, they are subject to degradation by nucleases and to dilution in replicating cells. In addition, many adenoviral contructs are cytotoxic or immunogenic, or interact with endogenous cellular signaling components. Thus, because of their complexities, viral vectors can pose a number of difficulties. That is not to say that viral vectors are not powerful and useful tools. Rather, they require a very high level of commitment and expertise that may be beyond the capacity of many laboratories.
The current work demonstrates an alternative method to increase the efficiency of transient transfection of plasmid-based expression vectors into cultured rat mesangial cells. The technique could be easily adapted to other cell types.2 Although access to a FACS instrument is absolutely required, special transfection methods or viral vectors are not needed. We acknowledge that other potential pitfalls of this method could be possible, including low overall yields of purified cells or potentially high hourly costs involved in obtaining the purified cells by sterile cell sorting. In addition, because the mesangial cells are transiently transfected with nonreplicating DNA that does not integrate into chromosomes, the purified cell populations cannot be expanded. Despite those potential limitations, this approach could currently be used effectively by many laboratories.
The principles of this method are straightforward. With the rapid quantitation of GFP fluorescence in living cells provided by FACS instrumentation, the transfection efficiency and assessment of cell-to-cell variability in expression level were simultaneously obtained in each experiment. We observed significant enrichment of mesangial cells that expressed a cotransfected target cDNA among cells selected for GFP fluorescence. Depending on experimental goals, the use of enriched populations of cells expressing the cDNA of interest should increase the signal-to-noise ratio of target protein assays. We also confirmed proper targeting, processing, and orientation of a membrane protein encoded by an expression vector. Transient transfection resulting in an appropriately processed gene product would enable physiologically relevant functional studies, eliminating the pitfall of possible compensatory changes associated with stable expression of the target cDNA.
There are several advantages to using GFP as a selection marker. Expression of GFP in mammalian cells does not typically cause significant biological consequences. Unlike other biomarkers, GFP does not require other exogenously applied cofactors, and can be easily detected in living cells. It is well suited for double-labeling studies in mesangial cells. For example, because PI has a different emission peak, we could use FACS to eliminate nonviable cells while concurrently enriching for GFP+ cells. In addition, the quantitative nature of the fluorescence profiles of the cell populations provided by FACS allows for adjustment of fluorescence intensity limits for cells sorting within each experiment. If obtaining a large number of cells is the primary consideration, the detection threshold for the GFP signal can be set at a low level. On the other hand, a higher threshold fluorescence value can be used if more efficient transfection is required for a particular application. This ability to "fine tune" the selection/sorting criteria distinguishes the technique described in this report from other strategies that require cotransfection protocols followed by a post hoc purification scheme. One example of this type of strategy is the pHOOK system from Invitrogen. In that system, cells are transfected with a cDNA that encodes a fusion protein of the transmembrane domain of the platelet-derived growth factor receptor and an extracellular single chain antibody (sFv) raised against the hapten, 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one. After transfection, cells are enriched by incubation with magnetic beads coated with this hapten (3).
The success of our procedure, and other marker methods, for selecting
transfected cells depends on the tendency for cells that take up the
marker DNA to take up the target DNA as well. Various markers other
than GFP have previously been used to track and monitor transfection
efficiency; in fact, -galactosidase is frequently used for this
purpose. In the absence of precise cell-to-cell quantitation of
expression, however, individual successfully transfected cells have
generally been assumed to express the marker and target proteins in
constant proportion (30). Our data indicate that this assumption is not
entirely valid. The efficiency of target gene expression
(
-galactosidase or NPR-C) roughly corresponded with the marker GFP
expression (Figs. 3 and 4). However, we observed, after cotransfection
with GFP and
-galactosidase, that 29.7% of unsorted cells displayed
a fluorescent signal, yet only 10% of the unsorted population stained
positive for
-galactosidase. Even after cell sorting, not all of the
GFP+ cells stained positive for
-galactosidase. Only
50% of them did so. The discrepancy between
the fraction of cells that show GFP signal and
-galactosidase
staining might be due to variability in the proportional level of
expression of the respective gene products among cells and/or
differential sensitivity of the methods used to detect the proteins.
Although we can comfortably conclude that
GFP+ cells are much more likely to
express the cotransfected target cDNAs than are the unsorted
transfectants or the GFP
cells, the correlation between marker and target gene expression appears to be substantially <100%.
This initial description of the application of GFP to select transfectants could easily be improved and broadened. Numerous investigators have previously ligated GFP cDNA on either end of the target cDNA insert in expression vectors to observe the intracellular distribution of their protein of interest. Translation of these constructs results in concatameric proteins in which the GFP is aligned end to end on the carboxyl or amino terminus of the target protein. This strategy has been made practical by the relatively small size of GFP, its rather silent nature in mammalian systems, and the availability of commercial vectors designed for easy in-frame construction and expression of GFP fusion proteins. The approach has been validated in numerous recent studies of intracellular protein trafficking and delivery (8, 19, 28). As long as the function of the protein of interest is not disrupted, FACS could be used to select functional transfectants directly. Alternatively, vectors could be constructed that drive the expression of GFP and the target gene from separate open reading frames within the same DNA molecule. Another possible method to enhance our approach would be to use fluorescent nucleotide "paints", which have become widely available recently. Any of those methods could presumably result in a near-100% correlation between GFP and target gene expression in individual cells. Furthermore, cotransfection experiments could be envisioned with either technique, using multiple autofluorescent protein derivatives of GFP with emission spectra that do not overlap. Mutant green-, blue-, and gold-emitting versions of GFP have recently been marketed by Packard BioScience (Meriden, CT), Clontech, and Quantum Biotechnologies (Laval, Quebec, Canada).
In summary, we have described a method to obtain enriched populations of transiently transfected cells derived from early passage primary cultures of mesangial cells, a cell type that has previously been noted to be quite resistant to transfection. The required reagents are readily available to most laboratories, although FACS facilities may be less accessible. The efficiency of target protein expression in selected cells remains substantially <100%, but further improvements in the method can easily be envisioned. Even in its present form, the improvement in transfection efficiency obtainable with this method should facilitate applications such as reporter vector assays, or bulk expression of epitope-tagged signaling proteins for biochemical or cell biological assays.
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ACKNOWLEDGEMENTS |
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This work was supported by grants from the Department of Veterans Affairs (Merit Award to J. R. Raymond), the National Institutes of Health (Grants DK-52448 and HL-03710, to J. R. Raymond and E. L. Greene), Dialysis Clinics Inc. (to R. V. Paul), the South Carolina Affiliate of the American Heart Association (R. V. Paul and J. S. Grewal), and a laboratory endowment jointly supported by the MUSC Division of Nephrology and Dialysis Clinics Inc. (to J. R. Raymond) and MUSC University Research Foundation Awards (to M. N. Garnovskaya and E. L. Greene).
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
2 This method has also proven successful in primary cultures of rat vascular smooth muscle cells (X. Gong, G. Collinsworth, E. Greene, B. Egan, and J. R. Raymond; unpublished observations).
1 pEGFP-N series of NH2-terminal protein fusion vectors or pEGFP-C series of COOH-terminal protein fusion vectors are available from Clontech (Palo Alto, CA).
Address for reprint requests and other correspondence: J. R. Raymond, Rm. 829 Clinical Science Bldg., 171 Ashley Ave., Charleston, SC 29425-2227 (E-mail: raymondj{at}musc.edu).
Received 22 September 1998; accepted in final form 22 January 1999.
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Behr, J.-P.,
B. Demeneix,
J.-P. Loeffler,
and
J. Perez-Mutul.
Efficient gene transfer into mammalian primary endocrine cells with lipopolyamine-coated DNA.
Proc. Natl. Acad. Sci. USA
86:
6982-6986,
1989[Abstract].
2.
Biamonti, G.,
G. Della Valle,
D. Talarico,
F. Cobianchi,
S. Riva,
and
A. Falaschi.
Fate of exogenous recombinant plasmids introduced into mouse and human cells.
Nucleic Acids Res.
13:
5545-5561,
1985[Abstract].
3.
Chestnut, J. D.,
A. R. Baytan,
M. Russell,
M.-P. Chang,
A. Bernard,
I. H. Maxwell,
and
J. P. Hoeffler.
Selective isolation of transiently transfected cells from a mammalian cell population with vectors expressing a membrane-anchored single chain antibody.
J. Immunol. Methods
193:
17-27,
1997.
4.
Chuprun, J. K.,
J. R. Raymond,
and
P. J. Blackshear.
Heterotrimeric G protein Gi2 mediates LPA-stimulated mitogenic signalling in mouse fibroblasts.
J. Biol. Chem.
272:
773-780,
1997
5.
Cooper, M.,
M. Lippa,
J. M. Payne,
G. Hatzivassiliou,
E. Reifenberg,
B. Fayazi,
J. C. Perales,
L. J. Morrison,
D. Templeton,
R. L. Piekarz,
and
J. Tan.
Safety-modified episomal vectors for human gene therapy.
Proc. Natl. Acad. Sci. USA
94:
6450-6455,
1997
6.
Cubitt, A. B.,
R. Heim,
S. R. Adams,
A. E. Boyd,
L. A. Gross,
and
R. Y. Tsien.
Understanding and using green fluorescent proteins.
Trends Biochem. Sci.
20:
448-455,
1995[Medline].
7.
Dremier, S.,
V. Pohl,
C. Poteet-Smith,
P. P. Roger,
J. Corbin,
S. O. Doskeland,
J. E. Dumont,
and
C. Maenhaut.
Activation of cyclic AMP-dependent kinase is required but may not be sufficient to mimic cyclic AMP-dependent DNA synthesis and thyroglobulin expression in dog thyroid cells.
Mol. Cell. Biol.
17:
6717-6726,
1998[Abstract].
8.
Elliott, G.,
and
P. O'Hare.
Intercellular trafficking and protein delivery by a herpesvirus structural protein.
Cell
88:
223-233,
1997[Medline].
9.
Felgner, P.,
T. Gadek,
M. Holm,
R. Roman,
H. Chan,
M. Wenz,
J. Northrop,
G. Ringold,
and
M. Danielsen.
Lipofectin: a highly efficient lipid-mediated DNA-transfection procedure.
Proc. Natl. Acad. Sci. USA
84:
7413-7417,
1987[Abstract].
10.
Garnovskaya, M. N., T. W. Gettys, T. van
Biesen, V. Prpic, J. K. Chuprun, and J. R. Raymond.
5-HT1A receptor activates
Na+/H+
exchange in CHO-K1 cells through
Gi2 and
Gi
3. J. Biol.
Chem. 272: 7770-7776.
11.
Goff, S. P.
Integration of retroviral DNA into the genome of the infected cell.
Cancer Cells
2:
172-178,
1990[Medline].
12.
Graham, F. L.,
and
A. J. Van der Eb.
A new technique for the assay of infectivity of human adenovirus 5 DNA.
Virology
52:
456-467,
1973[Medline].
13.
Green, N. K.,
J. A. Franklyn,
V. Ohanian,
A. M. Heagerty,
and
M. D. Gammage.
Transfection of cardiac muscle: effects of overexpression of c-myc and c-fos proto-oncogene proteins in primary cultures of neonatal rat cardiac myocytes.
Clin. Sci. (Colch.)
92:
181-188,
1997[Medline].
14.
Guo, Z.,
N. S. Yang,
S. Jiao,
J. Sun,
L. Cheng,
J. A. Wolff,
and
I. D. Duncan.
Efficient and sustained transgene expression in mature rat oligodendrocytes in primary culture.
J. Neurosci. Res.
43:
32-41,
1996[Medline].
15.
Haas, J.,
E. C. Park,
and
B. Seed.
Codon usage limitation in the expression of HIV-1 envelope glycoprotein.
Curr. Biol.
6:
315-324,
1996[Medline].
16.
Heim, R.,
A. B. Cubitt,
and
R. Y. Tsien.
Improved green fluorescence.
Nature
373:
663-664,
1995[Medline].
17.
Imai, E.,
and
Y. Isaka.
Strategies of gene transfer to the kidney.
Kidney Int.
53:
264-272,
1998[Medline].
18.
Kaneda, Y.,
R. Morishita,
and
N. Tomita.
Increased expression of DNA cointroduced with nuclear protein in adult rat liver.
J. Mol. Med.
73:
289-297,
1995[Medline].
19.
Köhler, R. H.,
J. Cao,
W. R. Zipfel,
W. W. Webb,
and
M. R. Hanson.
Exchange of protein molecules through connections between higher plant plastids.
Science
276:
2039-2042,
1997
20.
Mahvi, D. M.,
J. K. Burkholder,
J. Turner,
J. Culp,
J. S. Malter,
P. M. Sondel,
and
N. S. Yang.
Particle-mediated gene transfer of granulocyte-macrophage colony-stimulating factor cDNA to tumor cells: implications for a clinically relevant tumor vaccine.
Hum. Gene Ther.
7:
1535-1543,
1996[Medline].
21.
Moritz, T.,
P. Dutt,
X. Xiao,
D. Carstanjen,
T. Vik,
H. Hanenberg,
and
D. A. Williams.
Fibronectin improves transduction of reconstituting hematopoietic stem cells by retroviral vectors: evidence of direct viral binding to chymotryptic carboxy-terminal fragments.
Blood
88:
855-862,
1996
22.
Muramatsu, T.,
Y. Mizutani,
Y. Ohmori,
and
J. Okumura.
Comparison of three nonviral transfection methods for foreign gene expression in early chicken embryos in ovo.
Biochem. Biophys. Res. Commun.
230:
376-380,
1997[Medline].
23.
Paul, R. V.,
P. S. Wackym,
M. Budisavljevic,
E. Everett,
and
J. S. Norris.
Regulation of atrial natriuretic peptide clearance receptor in mesangial cells by growth factors.
J. Biol. Chem.
268:
18205-18212,
1993
24.
Paul, R. V.,
P. S. Wackym,
and
M. Budisavljevic.
Destabilization of natriuretic peptide C-receptor mRNA by phorbol myristate acetate.
J. Am. Soc. Nephrol.
9:
26-32,
1998[Abstract].
25.
Porter, J. G.,
A. Arfsten,
F. Fuller,
J. A. Miller,
L. C. Gregory,
and
J. A. Lewicki.
Isolation and functional expression of the human atrial natriuretic peptide clearance receptor cDNA.
Biochem. Biophys. Res. Commun.
171:
796-803,
1988.
26.
Prasher, D. C.,
V. K. Eckenrode,
W. W. Ward,
F. G. Prendergrast,
and
M. J. Cormier.
Primary structure of the Aequorea victoria green-fluorescent protein.
Gene
111:
229-233,
1992[Medline].
27.
Sanes, J. R.,
J. L. R. Ruberstein,
and
J.-F. Nicolas.
Use of a recombinant retrovirus to study post-implantation cell lineage in mouse embryos.
EMBO J.
5:
3133-3142,
1986[Abstract].
28.
Shields, J. M.,
and
V. W. Yang.
Two potent nuclear localization signals in the gut-enriched Kruppel-like factor define a subfamily of closely related Kruppel proteins.
J. Biol. Chem.
272:
18504-18507,
1997
29.
Vaheri, A.,
and
J. S. Pagano.
Infectious poliovirus RNA: a sensitive method of assay.
Virology
27:
434-436,
1965[Medline].
30.
Wigler, M. R.,
G. K. Sweet,
B. Sim,
A. Wold,
E. Pellicer,
T. Lacy,
S. Maniatis,
R. Silverstein,
and
R. Axel.
Transformation of mammalian cells with genes from prokaryotes and eukaryotes.
Cell
16:
777-785,
1979[Medline].
31.
Zhou, X. H.,
A. L. Klibanov,
and
L. Huang.
Lipophilic polylysines mediate efficient DNA transfection in mammalian cells.
Biochim. Biophys. Acta
1065:
8-14,
1991[Medline].