Dicistronic selection for nuclear proteins in living animal cells

Dicistronic selection for nuclear proteins in living animal cells

Gene, 137(1993)145-149 0 1993 Elsevier Science Publishers B.V. All rights reserved. 145 0378-l 119/93/$06.00 GENE 07507 Dicistronic selection for...

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Gene, 137(1993)145-149 0 1993 Elsevier Science Publishers

B.V. All rights reserved.

145

0378-l 119/93/$06.00

GENE 07507

Dicistronic selection for nuclear proteins in living animal cells (Transcription;

anti-CD4 beads; magnetic field; myb; chicken monoblasts)

Jan Smarda and Joseph S. Lipsick Department of Microbiology, SUNYat

Stony Brook, Stony Brook, NY11794-5222,

Received by A.-M. Skalka: 4 May 1993; Revised/Accepted:

USA

19 July/20 July 1993; Received at publishers:

12 August

1993

SUMMARY

We present an expression/selection system designed for the purification of cell lines inducibly expressing genes coding for unselectable proteins by using dicistronic selection for the cell surface marker CD4. This system enabled us to establish and purify c-myb expressing variants of the v-myb transformed chicken monoblast cell line BM2 with high efficiency.

INTRODUCTION

The disadvantage of many transfection techniques commonly used for the introduction of exogenous genes into animal cells is their relatively low efficiency. Therefore transfected cells have to be analyzed together with or purified away from a large pool of untransfected cells. If the gene of interest does not provide the cells with a selectable phenotype, as in the case of many nuclear transcription factors, the problem of their selection is even more difficult because most commonly used selectable markers such as neo (Colbere-Garapin et al., 1982; Southern and Berg, 1982), Ecogpt (Mulligan and Berg, 1981) or HyR (Blochlinger and Diggelmann, 1984; Santerre et al., 1984) are enzymatic and display a threshCorrespondence to: Dr. J.S. Lipsick

at his present

address:

Department

of Pathology, Stanford University School of Medicine, Stanford, 94305-5324, USA. Tel. (1-415) 723-1623; Fax (1-415) 725-6902. Abbreviations:

AMP,

ampicillin;

bp, base pair(s); CD4, human

CA

cluster

of differentiation antigen 4 protein; CD4, gene encoding CD4; DMEM, Dulbecco-modified Eagle’s medium; Ecogpt, Escherichia coli gene coding for xanthine-guanine phosphoribosyltransferase; FACS, fluorescence-activated cell sorter; Jfy, hygromycin; IRES, internal ribosomal entry site; mAb, monoclonal antibody(ies); MT, metallothionein; neo, neomycin; ori, origin of DNA replication; PAGE, polyacrylamide-gel electrophoresis; polyA, polyadenylation signal; a, resistant/resistance; RBS, ribosome-binding site(s); SDS, sodium dodecyl sulfate; [I, denotes plasmid carrier state.

old of resistance rather than a strict dose dependence. In particular, construction of cell lines with inducible promoters often requires the analysis of many individual stable transfectants in order to identify clones with low basal levels and high induced levels of gene expression. We now describe an expression/selection system based on an inducible promoter driving the transcription of a two-gene element, consisting of the gene of interest followed by the internal ribosomal entry site (IRES) of encephalomyocarditis virus and a second gene coding for the CD4 protein. Both genes are transcribed as one unit giving rise to a dicistronic mRNA (Jang et al., 1989; Jang and Wimmer, 1990; Ghattas et al., 1991). The presence of two RBS within a single mRNA ensures that both proteins are synthesized independently, but in a coupled manner. The CD4 protein marks the surface of transfected cells which can thus be targeted with antiCD4 antibody-coated magnetic beads and purified by magnetic field. A significant enrichment of the unselected nuclear protein is detectable in the CDCpositive cell fraction. This system provides a tool to specifically target and purify clones with low uninduced basal levels and high induced levels of expression of the transfected gene by a two step selection (Fig. 1). First the cells expressing the desired gene constitutively are eliminated by selecting against CD4 expression in the uninduced cell pool (negative selection). Then, following induction, the cells induci-

146

Zn2’

IRES

00

i

CDd / c-Myd cell

@I

CD: / c-Myb+ cell

@,

anti-CD4 ferromagnetic bead

@@[email protected]@@@@ INDUCER

I

3

, magnet

,x DISCARD

CLONES

Fig. 1. Scheme for negative-positive selection of transfected cells inducibly expressing a gene of interest. First, cells constitutively expressing the dicistronically coupled CD4 and c-myb genes are negatively selected using anti-CD4 magnetic beads. Second, the MT promoter-driven dicistronic expression unit is induced by adding ZnCl, to the culture medium. Third, cells in which CD4 and c-myb expression has been induced are positively selected using anti-CD4 magnetic beads.

bly expressing CD4 are selected (positive selection). We have successfully used this system for establishing variants of the v-myb transformed chicken monoblast cell line BM2 which inducibly express c-myb.

EXPERIMENTAL AND DISCUSSION

(a) The elements of plasmid pMT-IRES-CD4 The pMT-IRES-CD4 plasmid (Fig. 2) contains the MT II* promoter (Karin et al. 1987), unique XbaI and ClaI sites suitable for cloning the gene of interest downstream from the promoter, an encephalomyocarditis virusderived IRES (Jang et al., 1989), a cDNA encoding human CD4 (Shaw et al., 1989) and an SV40-derived intron and polyadenylation site. To test the usefulness of this system we have cloned a chicken c-myb cDNA (the XbaI fragment from NeoCCC; Grasser et al., 1991) or v-myb cDNA (the XbaI fragment from SA CLA12NCOdGE; Ibanez and Lipsick, 1988), both coding for nuclear proteins, into the XbaI site of the plasmid pMT-IRESCD4, thus making the plasmids pMTcMYBCD4 and pMT-vMYB-CD4, respectively.

(b) Transfected quail fibroblasts express both v-lc-myb and CD4 upon treatment with ZnCl, The plasmids pMT-cMYB-CD4 or pMT-vMYB-CD4 were transiently introduced into quail fibroblast cell line QT6 by the Casphosphate transfection method (Chen and Okayama, 1987; Ibanez and Lipsick, 1990). Cells were treated with ZnCl, for 24 h before the harvest to activate MT promoter-directed transcription. The synthesis of both Myb and CD4 proteins was tested by Western blotting and immunoprecipitation, respectively (Fig. 3). The c-Myb protein was significantly less stable than v-Myb in these fibroblasts. (c) Inducible myb-CD4 coexpression makes transfected cells treated with anti-CD4-coated magnetic beads selectable with a magnetic field In order to assay the efficiency of purification of myb expressing cells through the selection for CD4, we performed transient transfection experiments introducing either the pMT-cMYB-CD4 or the pMT-vMYB-CD4 plasmid into QT6 cells. ZnCl,-treated cells were incubated with the anti-CD4 antibody-coated magnetic beads and fractionated with a magnetic field (magnetic particle concentrator, MPC-1, Dynal, Great Neck, NY, USA). The myb expression in cells of both magnetic and anti-

147

KpnI

Fig. 2. The map of plasmid pMT-IRESCD4. The human MTII, promoter is the HindIII-BamHI fragment of the plasmid pHS1 (Karin et al., 1987); the IRES element (from EcoRI to MscI) and the plasmid backbone are derived from the PBS-ECAT (Jang et al., 1989); CD4 cDNA (1521-bp NciI fragment) comes from the PBS-CD4 (Shaw et al., 1989). The start codon for CD4 is provided by the IRES element, the second triplet in the CD4 coding sequence is deleted and the third triplet is altered from CGG to GGG, thus coding for Gly instead of Arg. The remainder of the cDNA coding for CD4 has not been changed. A detailed description of pMT-IRES-CD4 construction is available upon request.

magnetic fractions was tested by Western blotting (Fig. 4). Most of the myb-expressing cells in both experiments were found in the ZnCl,-treated magnetic (CD43 fractions, suggesting that this system is suitable for the enrichment of cells expressing nuclear proteins, which are otherwise unselectable. Low but detectable presence of some Myb proteins in ZnCI,-treated antimagnetic fractions was observed even if an increased amount of beads was used. We believe this is a result of less efficient mixing of cells with the heavier beads which quickly fall to the bottom of the tube, thus some of the CD#-expressing cells escape the beads. This effect, however, does not interfere with the main goal of this system which is the enrichment of clones expressing unselectable nuclear proteins present in the CD4+ fraction. (d) Bead-mediated purification of the c-q&expressing BM2 cells We have successfully used the technique of negativepositive selection for the purification of stable c-~y~-CD~ transfectants and control CD4 transfectants of the v-mybtransformed chicken monoblast cell line BM2 (Moscovici et al., 1982). BM2 cells do not express detectable levels of endogenous c-~~~ but they do constitutively express the transforming oncogene v-myb, which encodes a truncated form of the c-Myb protein. In order to purify the BM2 clones expressing c-myb we have cotransfected

anti-Myb 1 2 3 4

anti-CD4 c

1

2

3

4

kDa

kL)a -c-Myb

6%

-v-Myb -c-Myb breakdown 43-

Zn-

-

+

-

+

Fig. 3. Coinducibility of myb and CD4 expression in transiently transfected quail fibroblasts. QT6 cells were transiently transfected with 3 pg of plasmids pMT-vMYB-CD4 (lanes 1, 2) or pMT-cMYB-CD4 (lanes 3, 4). The cells were either left without further treatment (lanes 1, 3) or treated for 24 h with ZnCl, (1 x 10e4M) (lanes 2, 4). Harvested cells were acetone precipitated (Lane et al., 1990) and samples with equal protein content were resolved by SDS-PAGE [ 10% PAGE (for Western blotting) or 7.5% (for immunopr~ipitation)]. The blot was probed with a mixture of mAb anti-Myb 2.2 and 2.7 (Evan et al., 1984). Blots were developed using rabbit anti-mouse immunoglobin G conjugated to alkaline phosphatase (Promega, Madison, WI, USA). Standard protocols were followed for [35S]methionine labeling of transfected cells and for the immunoprecipitation of CD4 protein with the monoclonal anti-CD4 antibody 0KT4 (ATCC) bound to protein A Sepharose via rabbit anti-mouse immunoglobin (Harlow and Lane, 1988). Lane C: Extract from CD4-expressing human CEM cells (positive control).

148 pMT-VMYB-CD4

pMT-cMYB-CD4

kDa I16845a48-

Zn

-

_

Fraction

AMAM

+

+

-

_

A

MAM

+

+

Fig. 4. Enrichment of myh-expressing cells via CD4 selection. Zn*+ -treated (1 x 10-“M) QT6 cells (3 x 107) transiently transfected with pMT-vMYBCD4 or pMT-cMYB-CD4 were washed with PBS and suspended with 2 ml of 1 mM solution of EDTA in PBS per 10 cm dish. 100 ul of DYNABEADS M-450 CD4 (Dynal, Norway) were added and slowly shaken for 15 min at 4°C. The cells were exposed to the magnetic field for 2 min, as recommended by the manufacturer and separated into CD4+ magnetic (M) and CD4- antima~etic (A) fractions. The pnyb expression in both fractions is shown in a Western blot (see Fig. 3 legend) probed with a mixture of the mAb anti-Myb 2.2 and 2.7 (Evan et al., 1984). PBS is 8 g NaC1/0.2 g KCl/0.24 g KH,P0,/1.44 Na,HPO,/lOOXl ml water, pH 7.4.

BM2 [ pMT-cMY B-CD4, pSV2Neol (Southern and Berg, 1982) by lipofection. 5 x lo6 cells were washed twice in serum-free OPTI-MEM media (Gibco BRL, Gaithersburg, MD, USA), resuspended in 3 ml of the same media, mixed with transfecting DNAs ( 15 ug of pMT-cMYB-CD4 and 3 p.g of pSV2Neo) and 30 ul of lipofectin (Gibco BRL) and incubated in a 5-cm plate for 2 h in a 37”C/lO% COz incubator. The reaction was stopped by addition of 3 ml DMEM and both fetal calf and chicken sera to concentrations of 5%. The third day after the transfection G418 (500 ug/ml) was added. The pool of G418-resistant cells was selected out in 3 weeks. To purify the BM2 clones inducibly expressing c-myb we have first selected against CD4 expression in the uninduced pMT-cMYB-CD4-transfected cell pool (Fig. 1); the cells which did not have affinity for anti-CD4 beads were then treated with ZnCl, to induce c-myb-CD4 expression and exposed to the beads again. The cells inducibly expressing CD4 were purified using a magnetic field. The level of Zn’+-dependent c-myb expression in these cells was tested by Western blotting (Fig. 5). A relatively sharp threshhold was observed. Clones were then isolated by limiting dilution in 96well plates. Use of bead purification increased the efficiency of isolation of inducible clones to 100%. We have also been able to rapidly isolate positive clones by micropipetting single positive cells identified by the formation of rosettes under phase contrast microscopy. To test the inducible, dicistronic character of c-mybCD4 transcripts we performed Northern blotting of total RNA purified from BM2cMYB cells and probed the blots either with c-myb or CD4 cDNAs. Both blots identified

kDa 1

2

3

4

5

6

c Cc-Myb

58Cv-Myb

[email protected]”QM)

1.5

0

.8

I

1.25 1.5

1

Fig. 5. Zn 2+ titration of BM2cMYB. BM2 (lane l), and BMZcMYB (lanes 2-6) cells were exposed to different concentrations of ZnCls for 24 h. A Western blot of cells extracts resolved by SDS-PAGE (see Fig. 3 legend) was probed with an anti-Myb 2.2 and 2.7 mAb mixture. The expression of c-myb in transiently transfected and %*+-treated QT6 cells is shown as a positive control (lane C).

mRNA molecules of the same expected size in ZnCl,treated BM2cMYB cells (Fig. 6), suggesting that both c-myb and CD4 sequences are present in the same mRNA molecules. In addition, as shown in Fig. 6, the c-myb probe also hybridized to endogenous 2-kb v-myb transcripts because of v-myb/c-myb homology. Lane 5 was somewhat underloaded. However, the 2-kb v-myb transcript was also somewhat Zn’+-inducible, presumably because of a response of the viral LTR to host-cell stress. We have described a unique expression/selection system which is useful for creating cell lines that inducibly express nuclear or other proteins lacking a selectable phenotype. The system provides both for inducibility of expression of a desired gene through the metallothionein promoter and for selection of transfected cells expressing the desired gene through the dicistronically coinduced

149 grants ROl CA 43592 and K04 CA 01457. Joseph S. Lipsick is a Scholar of the Leukemia Society of America.

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REFERENCES

C28S C18S

Zn

probe

-

+

-

CD4

+

-

+

-

+

vb

Fig. 6. Zn *+-inducible dicistronic c-myb transcription in v-myb transformed monoblasts. Total RNA (10 ug) purified from BM2 (lanes 1, 2, 5,6) and BM2cMYB (lanes 3,4,7,8) cells by the ~anidium isothiocyanate method, was resolved in a 1% agarose-2.2 M formaldehyde gel, transferred to a GeneScreen Plus membrane (DuPont NEN) and hybridized to 32P-labeled DNA probes. The myb probe was the c-myb cDNA fragment (XbaI) of the plasmid pMT-cMYB-CD4; the CD4 probe was the BamHFEcoRI fragment of the plasmid PBS-CD4 (Shaw et al., 1989). The RNA purification, transfer, probe labeling by the random primer method as well as washing of blots as described in Sambrook et al. (1989). To mark the position of 18s and 28s rRNAs, the RNA molecules on filter were stained with methylene blue (Sambrook et al., 1989).

cell surface marker CD4. The advantage of this system is that (XII-expressing cells can be gently purified with the anti-CD4 coated magnetic beads which do not decrease their viability. Alternatively, the FACS or antibody ‘panning’ techniques can be used for the enrichment of the CD&positive clones instead of using of anti-CD4 beads and a magnetic field. This system can be easily adopted for use with alternative inducible promoters and cell surface markers to permit independently inducible expression of multiple genes in a single cell. (e) Conclusions (I ) Dicistronic selection can be used to efficiently select for the co-expression of an otherwise unselectable gene in animal cells. (2) A negative-positive di~stronic selection scheme can be used to efficiently produce cell lines that inducibly express a desired gene product.

We thank Mike Bishop, Sun-Kee Jang, Michael Karin, Bart Sefton and Eckard Wimmer for supplying various clones and antibodies and Judy Nimmo for excellent secretarial assistance. This work was supported by USPHS

Blochlinger, K. and Di~elmann, H.: Hygromycin B phosphotransferase as a selectable marker for DNA transfer experiments with higher eukaryotic cells. Mol. Cell. Biol. 4 (1984) 2929-2932. Chen, C.H. and Okayama, H.: High-efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell. Biol. 7 (1987) 2745-2752. Colbere-Garapin, F., Horodniceanu, F., Kourilsky, P. and Garapin, A.-C.: A new dominant hybrid selective marker for higher eukaryotic cells. J. Mol. Biol. 150 (1982) l-14. Evan, G.I., Lewis, G.K. and Bishop, J.M.: Isolation of monoclonal antibodies specific for the products of the avian oncogene myb. Mol. Cell. Biol. 4 (1984) 2843-2850. Ghattas, I.R., Sanes, J.R. and Majors, J.E.: The encephalomyocarditis virus internal ribosome entry site allows efficient coexpression of two genes from a recombinant provirus in cultured cells and in embryos. Mol. Cell. Biol. 11 (1991) 5848-5859. Grasser, F.A., Graf, T. and Lipsick, J.S.: Protein truncation is required for the activation of the c-myb proto-oncogene. Mol. Cell. Biol. 11 (1991) 3987-3996. Harlow, E. and Lane, D.P.: Antibodies. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988. Ibanez, C.E. and Lipsick, J.S.: Structural and func~onal domains of the myb oncogene: requirements for nuclear transport, myeloid transformation, and colony formation. J. Virol. 62 (1988) 1981-1988. Ibanez, C.E. and Lipsick, J.S.: tram activation of gene expression by v-myb. Mol. Cell. Biol. 10 (1990) 2285-2293. Jang, S.K. and Wimmer, E.: Cap-independent translation of encephalomyocarditis virus RNA: structural elements of the internal ribosome entry site, and involvement of a cellular 57 KDa RNA-bin~ng protein. Genes Devel. 4 (1990) 1560-1572. Jang, S.K., Davies, M.V., Kaufman, R.J. and Wimmer, E.: Initiation of protein synthesis by internal entry of ribosomes into the 5’ nontranslated region of encephalomyocarditis virus RNA in vivo. J. Virol. 63 (1989) 1651-1660. Karin, M., Haslinger, A., Heguy, A., Dietlin, T. and Cooke, T.: Metalresponsive elements act as positive modulators of human metallothionein-11, enhancer activity. Mol. Cell. Biol. 7 (1987) 606-613. Lane, T.N., Ibanez, C.E., Garcia, A., Graf, T. and Lipsick, J.S.: Transformation by v-myb correlates with transactivation of gene expression. Mol. Cell. Biol. 10 (1990) 2591-2598. Moscovici, C., Zcller, N. and Moscovici, M.G.: Continuous lines of AMV-tranformed non-producer cells: growth and oncogenic potential in the chick embryo. In: Revoltella, R.F., Basilica, C., Gallo, R.C., Pontieri, G.M., Rovera, G. and Subak-Sharpe, J.H. (Eds.), Expression of Differentiated Function in Cancer Cells. Raven Press, New York, 1982, pp. 325-449. Mulligan, R.C. and Berg, P.: Selection for animal cells that express the Escherichia coli gene coding for xanthine-guanine phosphoribosyltransferase. Proc. Natl. Acad. Sci. USA 78 (1981) 2072-2076. Sambrook, J., Fritsch, E.F. and Maniatis, T.: Molecular Cloning. A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. Santerre, R.F., Allen, N.E., Hobbs Jr., J.N., Rao, R.N. and Schmidt, R.J.: Expression of prokaryotic genes for hygromycin B and G418 resistance as dominant-selection markers in mouse L cells. Gene 30 (1984) 147-156. Shaw, AS., Amrein, K.A., Hammond, C., Stern, D.F., Se&on, B.M. and Rose, J.K.: The lck protein kinase interacts with the cytoplasmic tail of the CD4 protein through its unique amino-terminal domain. Cell 59 (1989) 627-636. Southern, P.J. and Berg, P.: Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early gene promoter. J. Mol. Appl. Genet. 1 (1982) 327.--341.