Skip Navigation

This Article
Right arrow Abstract Freely available
Right arrow Print PDF (159K) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (7)
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Klucher, K. M.
Right arrow Articles by Daley, G. Q.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Klucher, K. M.
Right arrow Articles by Daley, G. Q.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© 1997 Oxford University Press 4858-4860

A novel method to isolate cells with conditional gene expression using fluorescence activated cell sorting (FACS)

A novel method to isolate cells with conditional gene expression using fluorescence activated cell sorting (FACS) Kevin M. Klucher, Melissa J. Gerlach and George Q. Daley*

Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USA

Received July 25, 1997; Revised and Accepted October 17, 1997

ABSTRACT

An inducible expression system using control elements of the tetracycline resistance operon has recently shown promise for conditional gene expression of any gene of interest. However, intensive screening of multiple independent clones is often required to find cell lines with optimal induction characteristics. By coupling expression of the gene of interest with a fluorescent marker, we have developed a novel fluorescence activated cell sorting (FACS) based strategy to isolate cells with desirable expression characteristics, thus alleviating the laborious isolation and analysis of multiple independent clones.

An inducible expression system using the control elements of the tetracycline resistance operon encoded in Tn10 of Escherichia coli has been increasingly used for tight control of gene expression in mammalian cell culture (1 ) and in transgenic animals (2 ). Briefly, a tetracycline-controlled transactivator protein made by fusing the tet repressor with the activation domain of herpes simplex virus VP16 stimulates transcription from a promoter containing a heptamer of tet-operator sequences directly upstream of a minimal promoter. A recent modification of this system involved mutagenesis of the tet repressor to allow binding to the tet operator only in the presence of tetracycline or derivatives such as doxycycline. This modified transactivator protein was termed the reverse tet-transactivator (3 ).

We have explored the use of the Tet system to develop conditional expression of the human oncogene BCR/ABL. Early in our efforts, we observed highly variable basal and induced levels of expression among independent clones, a problem inherent in conditional gene regulatory systems. By coupling expression of the gene of interest with a fluorescent marker, we have developed a fluorescence activated cell sorting (FACS) based strategy to isolate cells with desirable expression characteristics, thus alleviating the laborious isolation and analysis of multiple independent clones.

The plasmid pTeti[beta]geo was constructed to select for inducible gene expression with defined expression levels. The salient characteristic of this vector is the presence of a fusion gene, [beta]geo, encoding both a drug resistance (neomycin) and enzymatic marker ([beta]-galactosidase), linked to the tet-responsive promoter by an internal ribosomal entry site (IRES), sequences derived from picornaviruses that allow translation of bicistronic messenger RNAs (4 ). pTeti[beta]geo was constructed by the insertion of an XbaI fragment from pIRES-[beta]geo (5 ) containing the complete IRES-[beta]geo gene into the XbaI site present in the polylinker of the tet-responsive promoter plasmid, pUHD10-3 (6 ). A unique SacII cloning site is present between the tet-responsive promoter and the IRES-[beta]geo gene. For purposes of testing this system, a cDNA encoding BCR/ABL (7 ) was introduced into pTeti[beta]geo at the SacII site forming pTetP210i[beta]geo (Fig. 1 ).


Figure 1. Model for conditional BCR/ABL expression using pTetP210i[beta]geo.

BaF3 cells, a hematopoietic cell line dependent on the cytokine interleukin-3 (IL-3) for proliferation (8 ), were electroporated with both pUHG17-1 (3 ), encoding the reverse tet-transactivator, and a puromycin selectable marker. Selection in 2 µg/ml puromycin produced a population of BaF3 cells expressing the tetracycline-controlled transactivator protein. pTetP210i[beta]geo was introduced into these cells followed by selection for G418 resistance (at 2 mg/ml) in the presence of 1 µg/ml doxycycline. Selection for G418-resistant cells prior to FACS allowed for removal of cells which did not stably incorporate pTetP210i[beta]geo and those cells with the lowest induced levels of gene expression. G418-resistant cells were initially analyzed for [beta]-galactosidase activity in the presence or absence of doxycycline by FACS using the vital [beta]-galactosidase substrate, fluorescein di-[beta]-D-galactopyranoside (FDG) (9 ) (Fig. 2 A, left). Although the majority of uninduced cells had relatively low levels of [beta]-galactosidase activity, the range of expression varied over 20-30-fold. Induced cells had a 100-fold range in expression levels and, as in the uninduced population, a large number of cells expressed low levels of [beta]-galactosidase activity. The top 10% of the induced cells expressed [beta]-galactosidase at levels not present in the population of uninduced cells. To test the feasibility of isolating cells with the highest levels of induced expression, two rounds of sorting were carried out for induced cells with [beta]-galactosidase activity higher than 99% of uninduced cells in the population (positive sorting). Following two rounds of positive sorting, negative sorting was carried out on uninduced cells to remove cells with high basal levels of expression from the positively-sorted population. The FACS profile of sorted cells is shown in Figure 2 B (left). Uninduced cells show a uniformly low level of expression while the majority of the induced cells in the sorted population have levels of expression ~50-fold higher. This is likely an underestimate due to a limiting supply of FDG substrate in the induced cells. Induced cells with lower levels of [beta]-galactosidase activity were continually present within our population throughout sorting. Whether this is due to uneven loading of FDG or loss of inducibility in some of the sorted cells is not known. Western blot analysis using an antibody to ABL was used to directly compare [beta]-galactosidase activity with BCR/ABL expression. Densitometric quantitation of the western blot data indicated an ~10-fold increase in BCR/ABL expression with induction in the unsorted cells (Fig. 2 A, right). Following three rounds of sorting, BCR/ABL expression was induced at least 150-fold (Fig. 2 B, right).


Figure 2. Expression analysis of unsorted (A) and sorted (B) BaF3 cells containing pTetP210i[beta]geo. FACS analysis of [beta]-galactosidase activity is shown at left. Cells were grown with 10% FBS in RPMI supplemented with IL-3 (supplied as 10% conditioned medium from Wehi3B cells) in the absence (green) or presence (red) of 1 µg/ml doxycycline (Sigma). Cells were induced for 72 h prior to FDG staining. Cells were stained by prewarming 1 × 106 cells in 100 µl growth media at 37°C for 5 min prior to addition of 100 µl 2 mM FDG in H2O (Molecular Probes) prewarmed to 37°C. After addition of FDG, cells were placed at 37°C for 1 min and then directly to ice. An aliquot of 2 ml of cold growth media was added and cells were incubated on ice for 30 min prior to analysis on a FACScan unit (Becton-Dickinson). [beta]-galactosidase activity is represented on the x-axis as FL1 fluorescence units and the number of cells scoring for a given fluorescence is shown as counts on the y-axis. Staining for cell sorting was as above except cell concentration was increased 5-fold. Cells were sorted as described in the text using a FACStar Plus sorter (Becton-Dickinson). Western blot analysis of ABL protein expression is shown at right. Cells grown as above were lysed in ABL lysis buffer (1% Triton X-100, 10 mM Tris, pH 7.6, 5 mM EDTA, 50 mM NaCl, 30 mM NaPPi, 5 mM PMSF). An aliquot of 50 µg protein was run on a 6% acrylamide resolving gel, transferred to nitrocellulose, blocked in 10% milk in phosphate-buffered saline (PBS), 0.05% Tween-20, incubated with a rabbit polyclonal antibody to ABL o/n in 5% milk in PBS (10), 0.05% Tween-20. HRP-conjugated goat anti-rabbit IgG secondary antibody (Santa Cruz Biotech.) and ECL were used to detect both BCR/ABL and constitutive cABL protein. Densitometric analysis was performed using MacBas software (Fuji Photo Film Co.).


Figure 3. Effect of conditional BCR/ABL expression on IL-3-dependent cellular proliferation. Prior to proliferation assay, all cells were grown with 10% FBS in RPMI supplemented with 10% conditioned medium from Wehi3B cells (as a source of IL-3) in the absence or presence of 2 µg/ml doxycycline (Sigma). Cells were washed extensively in PBS to remove IL-3 and suspended at 2 × 105 cells/ml in 10% FBS in RPMI in the absence or presence of 4 µg/ml doxycycline. 1 x 104 cells were aliquotted in triplicate to 50 µl growth media containing varying amounts of IL-3 (supplied as conditioned medium from Wehi3B cells) and grown for 2 days at 37°C. An aliquot of 30 µl 5 mg/ml MTT dye (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Sigma) was added (1.2 mg/ml final) and incubated at 37°C for 5 h. Acidic isopropanol (100 µl; 0.1 N HCl) was added, mixed, and the solution was analyzed at OD 570 nm immediately. The relative concentration of IL-3 is shown on the x-axis with the maximum concentration of Wehi3B conditioned medium (3.66%) set as 1. Results were normalized by setting the absorbance (OD 570) at the maximum concentration of Wehi3B conditioned medium for each cell population at 1. The relative absorbance (shown on the y-axis) is directly related to the number of viable cells within a sample. The average and standard deviation from triplicate samples are shown. [squf], B210; -, sorted/+dox; s, unsorted/+dox; u, unsorted/-dox; [squ], sorted/-dox; [circle], BaF3.

Expression of BCR/ABL transforms factor-dependent BaF3 cells to factor-independence (10 ). This transformation assay was used to determine the effect of sorting on the biological activity of BCR/ABL. The response of unsorted and sorted cells to IL-3 dose was measured using an MTT proliferation assay (Fig. 3 ) (11 ). Constitutive expression of BCR/ABL in BaF3 cells (10 ) ([squf], B210) and induced cells in the sorted population (-, sorted/+dox) were able to proliferate without regard to IL-3 concentration. In contrast, parental BaF3 cells showed a dose-dependent IL-3 requirement for cell proliferation ([circle], BaF3) with all cells undergoing apoptosis within 2 days of IL-3 withdrawal. Uninduced cells from the sorted population also showed a dose-dependent requirement on IL-3 for cell proliferation ([squ], sorted/-dox), with equivalent levels of MTT incorporation as the parental BaF3 cells in the absence of IL-3. However, at higher levels of IL-3 a shift in the dose-response curve is apparent in this population of cells compared to parental BaF3 cells. This may be due to the basal level expression of BCR/ABL present in these cells producing hypersensitivity to IL-3 (12 ). Unsorted cells (s, unsorted/+dox,u, unsorted/-dox) showed intermediate IL-3 dependence, in agreement with the levels of expression seen by FACS and western blot analysis (Fig. 2 A). Although the above experiments were performed using a saturating level of doxycycline, variation in doxycycline concentration enables titration of protein expression and doxycycline-dependence of proliferation (unpublished data).

These results demonstrate the feasibility of using FACS to isolate cells with conditional [beta]-galactosidase expression and corresponding conditional expression of genes placed upstream of the IRES-[beta]geo gene. A key advantage of this system is the ability to easily isolate a population of cells consisting of multiple independent clones with a similar conditional expression pattern. This will limit non-specific biological effects due to clonal variation and loss of cell lines due to instability of the transactivator or inducible genes. If independent clones are desired this approach will limit the number of clones to be screened. In this report we initially used positive sorting of induced cells to isolate cells with the highest level of gene expression, followed by negative sorting of uninduced cells to remove cells with unacceptably high basal levels of expression. Alternatively, an initial negative sort of uninduced cells could be used to isolate cells with the lowest basal levels of expression present within the population, followed by positive sorting of induced cells to remove cells incapable of induction. This strategy could also be adopted for detection of other linked genes (green fluorescent protein, cell surface markers).

ACKNOWLEDGEMENTS

We gratefully acknowledge Prof. H. Bujard for Tet-expression system plasmids. We also thank G. Paradis for help in FACS and E. Koh for helpful discussion. This work was supported by a grant from the American Cancer Society (#IRG-173F). G.Q.D. is a recipient of a Burroughs Wellcome Fund Career Award in the Biomedical Sciences.

REFERENCES

1 Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89, 5547-5551. MEDLINE Abstract

2 Furth, P. A., St. Onge, L., Boeger, H., Gruss, P., Gossen, M., Kistner, A., Bujard, H. and Hennighausen, L. (1994) Proc. Natl. Acad. Sci. USA 91, 9302-9306. MEDLINE Abstract

3 Gossen, M., Freundlieb, S., Bender, G., Mueller, G., Hillen, W. and Bujard, H. (1995) Science 268, 1766-1769. MEDLINE Abstract

4 Jang, S. K., Krausslich, H.-G., Nicklin, M. J. H., Duke, G. M., Palmenberg, A. C. and Wimmer, E. (1988) J. Virol. 62, 2636-2643. MEDLINE Abstract

5 Mountford, P., Zevnik, B., Duwel, A., Nichols, J., Li, M., Dani, C., Robertson, K., Chambers, I. and Smith, A. (1994) Proc. Natl. Acad. Sci. USA 91, 4303-4307. MEDLINE Abstract

6 Resnitzky, D., Gossen, M., Bujard, H. and Reed, S. I. (1994) Mol. Cell. Biol. 14, 1669-1679. MEDLINE Abstract

7 Daley, G. Q., Van Etten, R. A. and Baltimore, D. (1990) Science 247, 824-830. MEDLINE Abstract

8 Palacios, R. and Steinmetz, M. (1985) Cell 41, 727-734. MEDLINE Abstract

9 Nolan, G. P., Fiering, S., Nicolas, J.-F. and Herzenberg, L. A. (1988) Proc. Natl. Acad. Sci. USA 85, 2603-2607. MEDLINE Abstract

10 Daley, G. Q. and Baltimore, D. (1988) Proc. Natl. Acad. Sci. USA 85, 9312-9316. MEDLINE Abstract

11 Mosmann, T. (1983) J. Immunol. Methods 65, 55-63. MEDLINE Abstract

12 Gishizky, M. L. and Witte, O. W. (1992) Science 256, 836-839. MEDLINE Abstract

*To whom correspondence should be addressed. Tel: +1 617 258 7209; Fax: +1 617 258 5578; Email: daley@wi.mit.edu
Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow Print PDF (159K) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (7)
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Klucher, K. M.
Right arrow Articles by Daley, G. Q.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Klucher, K. M.
Right arrow Articles by Daley, G. Q.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?