EXPERIMJZNTAL
CELL RESEARCH
197,229-233 (19%)
Blasticidin S-Resistance Gene (bsr): A Novel Selectable Marker for Mammalian Cells MASAKO IZLJMI,**~ HIROSHI MIYAZAWA,* TAKASHI KAMAKURA,~ ISAIW YAMAGUCHI,$ TOYOSHIGE
ENDO,$ AND FUMIO HANAOKA*+’
*Cellular Physiology and $Uicmbial Toxicology Laboratories, RIKEN Institute, Saitamu 351-01, Japan; tOchurwmizu University, Tokyo 112, Japan; and $Kyoritsu College of Pharmacy, Tokyo 105, Japan
called blastidic acid [2, 31. Blasticidin S specifically inhibits protein synthesis in both prokaryotes and eukaryotes [4] and is widely used as a fungicide for the prevention of rice blast disease in Japan, Korea, and South America. Bacillus cereus strain K55-Sl, which is resistant to blasticidin S, was found to produce blasticidin S deaminase [5]. This enzyme converts blasticidin S to an inactive deaminohydroxy derivative [5, 61. Kecently, the structural gene of blasticidin S deaminase, the bsr gene, was isolated from a plasmid of B. cereus K55-Sl [7,8]. A 617-bp fragment containing the coding region for blasticidin S deaminase and its promoter region conferred blasticidin S resistance on Escherichia coli and Bacillus subtilis when integrated into multicopy plasmid vectors [8] or integrated into the chromosome even as a single copy [9]. In this paper, we report that mammalian cells transfected with a vector that carries the bsr gene were resistant to blasticidin S. We also compared the bsr gene with the aminoglycoside phosphotransferase gene, the neo gene, which is the most widely used as a selectable marker.
Blasticidin S is a microbial antibiotic that inhibits protein synthesis in both prokaryotes and eukaryotes. The blasticidin S-resistance gene (bsr), isolated from Bacillus cereus KSS-Sl strain, was inserted into pSV2 plasmid vector and introduced into cultured mammalian celcls by transfection. The bsr gene was integrated into the genome and conferred blasticidin S resistance on HeLa cells. The transfection frequency of the bsr gene was as high as that of the aminoglycoside phosphotransferase gene, the so-called neo gene, which is a representative selectable marker for mammalian cells. Transfectants in which several copies of bar had been integrated into the genome were highly resistant to blasticidin S. Furthermore, blasticidin S killed the cells more rapidly than G418, which is conventionally used as a selective drug for the neo gene. Thus bsr is concluded to be useful as a drug-resistance marker for 0 lSS1 Academic. Press. Inc. mammalian cells.
INTRODUCTION
Introduction of DNA into cultured mammalian cells is now a common procedure for many purposes, such as expression cloning, overproduction of proteins, and identification of elements involved in the control of gene expression. In most cases, cell lines that express the transfected gene are identified by the introduction of a selectable marker that confers resistance to a certain selective drug. However, the number of dominantacting genetic markers available for use in any type of cell is limited. Therefore, the development of new dominant-acting genetic markers such as drug-resistance markers would be very useful. Blasticidin S is a nucleoside antibiotic produced by Streptomyces griseochromogenes [l] and has the structure shown in Fig. 1. Blasticidin S consists of a pyrimidine nucleoside called cytosinine and a B-amino acid i To whom correspondence and reprint requests should be addressed at Cellular Physiology Laboratory, RIKEN Institute, 2-l Hirosawa, Wako-shi, Saitama 351-01, Japan. Fax: 0484-62-4673.
MATERIALS
AND METHODS
Materials. The sources of materials used in this work were as follows: G418 from Sigma Chemical Co., Ltd.; all restriction enzymes except NM, T4 DNA ligase, Klenow fragment, and HindIII linker were from Takara Shuzo Co. Ltd; and N&I was from Nippon Gene Co., Ltd. Blasticidin S was kindly supplied by Kaken Pharmaceutical Co., Ltd. Reactions of enzymes were done as recommended by the suppliers. CeUculture and DNA tmnsfection. HeLa cells were maintained in minimal essential medium containing 10% calf serum. Transfectants were maintained in the same medium containing 2 ag/ml of blasticidin S or 400 pg/ml of G418. The cells were maintained at 37°C in a humidified atmosphere containing 5% COs. Samples of 5 X 106HeLa cells were seeded onto 100 mm dishes, and 24 h later, 5 or 20 cg of supercoiled plasmid DNA without carrier DNA was introduced into the cells by the calcium phosphate precipitation method as moditled by Chen and Okayama [lo]. After exposure to the plasmid DNA at 35°C for 24 h under 3% CO, in air, the cells were washed twice with phosphate-buffered saline, refed with 10 ml of fresh medium, and cultured for 48 h at 37’C in an incubator under 5% CO, in air. Then the cells were collected by trypsin treatment and
229
0014~4827/91$3.00 Copyright Q 1991 All rights of reproduction
by AcademicPress,Inc. in any form reserved.
230
IZUMI ET AL.
NH
NH,
0
FIG. 1. Structures of blasticidin S and deaminoblasticidin Blasticidin S, R=NH,; deaminoblasticidin S, R=OH.
S.
replated at 1:lO dilution, and after 24 h, blasticidin S or G418 was added to the medium. The medium containing the drug was changed every 4 days, and individual colonies were isolated after 14 days. The numbers of colonies were determined by fixing the remaining cells with methanol and then staining them with Giemsa solution. Southern and Northern blot hybridization analyses. High molecular weight cellular DNA was extracted as previously described [ll]. Samples of 10 wg of DNA were digested with EcoRI, and the digests were separated by electrophoresis in 0.8% agarose gel. After alkaline treatment, DNA was transferred to a Hybond-N membrane and hybridized with the azP-labeled bsr gene generated by random hexamer priming. Total RNA was extracted with guanidinium isothiocyanate and the extract was centrifuged in cesium chloride solution [12]. A sample of 30 pg of total RNA was separated by electrophoresis in 1.2% agarose gel containing formaldehyde, transferred to a Hybond-N membrane, and hybridized with the ‘sP-labeled bsr gene. The hybridizing solution contained 50% formamide, 2X SSC buffer (1X SSC: 0.15 M NaCl, 0.015 M Na-citrate), 2X Denhardt’s solution, 50 rg/ml of salmon sperm DNA, and agP-labeled probe. Hybridization was performed at 42’C for 16 h and then the membranes were washed twice with 2X SSC buffer at 68°C for 10 min, once with 1X SSC buffer and once with 0.5~ SSC buffer. Autoradiography was carried out at -80°C for 7 days with an intensifying screen. Construction of pSV2bsr2. Plasmid pSV2bsr2 expressing the bsr gene in mammalian cells was constructed as follows (Fig. 2). The bsr gene was subcloned as a 617 bp N&I fragment into pUC19 [9]. The
Concentration of Blastfcffin S or G418(pg/ml)
FIG. 3. Effects of blasticidin S and G418 on colony formation in HeLa cells. HeLa cells were plated onto 60-mm dishes and cultured in the presence of the indicated concentrations of blasticidin S (0) and G418 (0). Numbers of colonies were counted after 2 weeks culture.
617 bp N&I fragment containing the bsr gene and its promoter region was digested with Hi&I. The resulting 462 bp HinfI-Nc?eI fragment, which contains only the coding region of blasticidin S deaminase, was purified by electrophoresis and ligated to HindIII linker. pSV2nco [13] was digested with BamHI and HindIII, and the 3.4-kb HindIIIBamHI fragment, containing the SV40 early promoter, pBR322 ori, and ampicillin-resistant gene, was purified. pSV2neo was also digested with PuuII and BamHI, and the l-kb PuuII-BamHI fragment, which contains part of the neo gene and the polyadenylation signal, was isolated and ligated to HindIII linker at the PuuII site. The 3.4-kb HindIII-BamHI fragment of pSV2nc0, the 1-kb PuuII-BamHI fragment of pSV2ne0, and the 462-bp bsr fragment were ligated. The resulting plasmid, pSV26sr2, contained two copies of the bsr gene. Plasmids were purified by the alkali lysis method followed by equilibrium centrifugation in a CsCl gradient as described [14]. Colony formation assay. Samples of 2 X 10xto 10’ cells were plated onto 60 mm dishes and cultured in the presence of the indicated con-
H P
P F
N
w FIG. 2. Construction of pSV2bsr2. The 462-bp Hi&I-NdeI fragment containing the bsr gene was ligated to HindIII linker. pSV2neo was digested with HindIII and BarnHI. and the 3.4-kb HindHI-BamHI fragment was isolated. The PouII-BamHI fragment of pSV2neo was also isolated, and the Hind111 linker was ligated to the PuuII site. The 462 bp bsr fragment, the HindIII-BamHI fragment of pSV2ne0, and the PvuII-BamHI fragment of pSV2neo were ligated. The resulting plasmid was designated as pSV2bsr2. pSVSbsr2 contained two copies of bsr. B, BarnHI; E, EcoRI; F, Hinfl, H, HindHI; N, N&I; P, PuuI1; open box, nco gene; hatched box, bsr gene; dotted box, SV40 early promoter.
BLASTICIDIN
TABLE
S-RESISTANCE
1
12345678910
Plasmid
G418 (400 Irdd
Blasticidin (2 dml)
S
12345678
wF4 23-
Exp. 2
Exp. 1 Blasticidin S (2 kg/ml)
(8)
(4
Efficiency of Transfection”
231
MARKER
G418 (400 PImu
9.46.64.4-
NOIW psv2neo pSV2bw2
t1.3 x 10-E t1.3 x 10-B 1.2 x 10-8
cl.3 x 10” 1.5 x 10-a
CELL RESEARCH
197,229-233 (19%)
Blasticidin S-Resistance Gene (bsr): A Novel Selectable Marker for Mammalian Cells MASAKO IZLJMI,**~ HIROSHI MIYAZAWA,* TAKASHI KAMAKURA,~ ISAIW YAMAGUCHI,$ TOYOSHIGE
ENDO,$ AND FUMIO HANAOKA*+’
*Cellular Physiology and $Uicmbial Toxicology Laboratories, RIKEN Institute, Saitamu 351-01, Japan; tOchurwmizu University, Tokyo 112, Japan; and $Kyoritsu College of Pharmacy, Tokyo 105, Japan
called blastidic acid [2, 31. Blasticidin S specifically inhibits protein synthesis in both prokaryotes and eukaryotes [4] and is widely used as a fungicide for the prevention of rice blast disease in Japan, Korea, and South America. Bacillus cereus strain K55-Sl, which is resistant to blasticidin S, was found to produce blasticidin S deaminase [5]. This enzyme converts blasticidin S to an inactive deaminohydroxy derivative [5, 61. Kecently, the structural gene of blasticidin S deaminase, the bsr gene, was isolated from a plasmid of B. cereus K55-Sl [7,8]. A 617-bp fragment containing the coding region for blasticidin S deaminase and its promoter region conferred blasticidin S resistance on Escherichia coli and Bacillus subtilis when integrated into multicopy plasmid vectors [8] or integrated into the chromosome even as a single copy [9]. In this paper, we report that mammalian cells transfected with a vector that carries the bsr gene were resistant to blasticidin S. We also compared the bsr gene with the aminoglycoside phosphotransferase gene, the neo gene, which is the most widely used as a selectable marker.
Blasticidin S is a microbial antibiotic that inhibits protein synthesis in both prokaryotes and eukaryotes. The blasticidin S-resistance gene (bsr), isolated from Bacillus cereus KSS-Sl strain, was inserted into pSV2 plasmid vector and introduced into cultured mammalian celcls by transfection. The bsr gene was integrated into the genome and conferred blasticidin S resistance on HeLa cells. The transfection frequency of the bsr gene was as high as that of the aminoglycoside phosphotransferase gene, the so-called neo gene, which is a representative selectable marker for mammalian cells. Transfectants in which several copies of bar had been integrated into the genome were highly resistant to blasticidin S. Furthermore, blasticidin S killed the cells more rapidly than G418, which is conventionally used as a selective drug for the neo gene. Thus bsr is concluded to be useful as a drug-resistance marker for 0 lSS1 Academic. Press. Inc. mammalian cells.
INTRODUCTION
Introduction of DNA into cultured mammalian cells is now a common procedure for many purposes, such as expression cloning, overproduction of proteins, and identification of elements involved in the control of gene expression. In most cases, cell lines that express the transfected gene are identified by the introduction of a selectable marker that confers resistance to a certain selective drug. However, the number of dominantacting genetic markers available for use in any type of cell is limited. Therefore, the development of new dominant-acting genetic markers such as drug-resistance markers would be very useful. Blasticidin S is a nucleoside antibiotic produced by Streptomyces griseochromogenes [l] and has the structure shown in Fig. 1. Blasticidin S consists of a pyrimidine nucleoside called cytosinine and a B-amino acid i To whom correspondence and reprint requests should be addressed at Cellular Physiology Laboratory, RIKEN Institute, 2-l Hirosawa, Wako-shi, Saitama 351-01, Japan. Fax: 0484-62-4673.
MATERIALS
AND METHODS
Materials. The sources of materials used in this work were as follows: G418 from Sigma Chemical Co., Ltd.; all restriction enzymes except NM, T4 DNA ligase, Klenow fragment, and HindIII linker were from Takara Shuzo Co. Ltd; and N&I was from Nippon Gene Co., Ltd. Blasticidin S was kindly supplied by Kaken Pharmaceutical Co., Ltd. Reactions of enzymes were done as recommended by the suppliers. CeUculture and DNA tmnsfection. HeLa cells were maintained in minimal essential medium containing 10% calf serum. Transfectants were maintained in the same medium containing 2 ag/ml of blasticidin S or 400 pg/ml of G418. The cells were maintained at 37°C in a humidified atmosphere containing 5% COs. Samples of 5 X 106HeLa cells were seeded onto 100 mm dishes, and 24 h later, 5 or 20 cg of supercoiled plasmid DNA without carrier DNA was introduced into the cells by the calcium phosphate precipitation method as moditled by Chen and Okayama [lo]. After exposure to the plasmid DNA at 35°C for 24 h under 3% CO, in air, the cells were washed twice with phosphate-buffered saline, refed with 10 ml of fresh medium, and cultured for 48 h at 37’C in an incubator under 5% CO, in air. Then the cells were collected by trypsin treatment and
229
0014~4827/91$3.00 Copyright Q 1991 All rights of reproduction
by AcademicPress,Inc. in any form reserved.
230
IZUMI ET AL.
NH
NH,
0
FIG. 1. Structures of blasticidin S and deaminoblasticidin Blasticidin S, R=NH,; deaminoblasticidin S, R=OH.
S.
replated at 1:lO dilution, and after 24 h, blasticidin S or G418 was added to the medium. The medium containing the drug was changed every 4 days, and individual colonies were isolated after 14 days. The numbers of colonies were determined by fixing the remaining cells with methanol and then staining them with Giemsa solution. Southern and Northern blot hybridization analyses. High molecular weight cellular DNA was extracted as previously described [ll]. Samples of 10 wg of DNA were digested with EcoRI, and the digests were separated by electrophoresis in 0.8% agarose gel. After alkaline treatment, DNA was transferred to a Hybond-N membrane and hybridized with the azP-labeled bsr gene generated by random hexamer priming. Total RNA was extracted with guanidinium isothiocyanate and the extract was centrifuged in cesium chloride solution [12]. A sample of 30 pg of total RNA was separated by electrophoresis in 1.2% agarose gel containing formaldehyde, transferred to a Hybond-N membrane, and hybridized with the ‘sP-labeled bsr gene. The hybridizing solution contained 50% formamide, 2X SSC buffer (1X SSC: 0.15 M NaCl, 0.015 M Na-citrate), 2X Denhardt’s solution, 50 rg/ml of salmon sperm DNA, and agP-labeled probe. Hybridization was performed at 42’C for 16 h and then the membranes were washed twice with 2X SSC buffer at 68°C for 10 min, once with 1X SSC buffer and once with 0.5~ SSC buffer. Autoradiography was carried out at -80°C for 7 days with an intensifying screen. Construction of pSV2bsr2. Plasmid pSV2bsr2 expressing the bsr gene in mammalian cells was constructed as follows (Fig. 2). The bsr gene was subcloned as a 617 bp N&I fragment into pUC19 [9]. The
Concentration of Blastfcffin S or G418(pg/ml)
FIG. 3. Effects of blasticidin S and G418 on colony formation in HeLa cells. HeLa cells were plated onto 60-mm dishes and cultured in the presence of the indicated concentrations of blasticidin S (0) and G418 (0). Numbers of colonies were counted after 2 weeks culture.
617 bp N&I fragment containing the bsr gene and its promoter region was digested with Hi&I. The resulting 462 bp HinfI-Nc?eI fragment, which contains only the coding region of blasticidin S deaminase, was purified by electrophoresis and ligated to HindIII linker. pSV2nco [13] was digested with BamHI and HindIII, and the 3.4-kb HindIIIBamHI fragment, containing the SV40 early promoter, pBR322 ori, and ampicillin-resistant gene, was purified. pSV2neo was also digested with PuuII and BamHI, and the l-kb PuuII-BamHI fragment, which contains part of the neo gene and the polyadenylation signal, was isolated and ligated to HindIII linker at the PuuII site. The 3.4-kb HindIII-BamHI fragment of pSV2nc0, the 1-kb PuuII-BamHI fragment of pSV2ne0, and the 462-bp bsr fragment were ligated. The resulting plasmid, pSV26sr2, contained two copies of the bsr gene. Plasmids were purified by the alkali lysis method followed by equilibrium centrifugation in a CsCl gradient as described [14]. Colony formation assay. Samples of 2 X 10xto 10’ cells were plated onto 60 mm dishes and cultured in the presence of the indicated con-
H P
P F
N
w FIG. 2. Construction of pSV2bsr2. The 462-bp Hi&I-NdeI fragment containing the bsr gene was ligated to HindIII linker. pSV2neo was digested with HindIII and BarnHI. and the 3.4-kb HindHI-BamHI fragment was isolated. The PouII-BamHI fragment of pSV2neo was also isolated, and the Hind111 linker was ligated to the PuuII site. The 462 bp bsr fragment, the HindIII-BamHI fragment of pSV2ne0, and the PvuII-BamHI fragment of pSV2neo were ligated. The resulting plasmid was designated as pSV2bsr2. pSVSbsr2 contained two copies of bsr. B, BarnHI; E, EcoRI; F, Hinfl, H, HindHI; N, N&I; P, PuuI1; open box, nco gene; hatched box, bsr gene; dotted box, SV40 early promoter.
BLASTICIDIN
TABLE
S-RESISTANCE
1
12345678910
Plasmid
G418 (400 Irdd
Blasticidin (2 dml)
S
12345678
wF4 23-
Exp. 2
Exp. 1 Blasticidin S (2 kg/ml)
(8)
(4
Efficiency of Transfection”
231
MARKER
G418 (400 PImu
9.46.64.4-
NOIW psv2neo pSV2bw2
t1.3 x 10-E t1.3 x 10-B 1.2 x 10-8
cl.3 x 10” 1.5 x 10-a
