The transcription of the a-globin and DHFR genes increased as their copy number within ..... transcription was inhibited in cell lines containing these genes after ...
Vol. 4. No. 8
MOLECULAR AND CELLULAR BIOLOGY. Aug. 1984, p. 1469-1475 0270-7306/84/081469-07$02.00/0 Copyright C) 1984, American Society for Microbiology
Amplification and Expression of Human o.-Globin Genes in Chinese Hamster Ovary Cells YUN-FAI LAU,* C. C. LIN,t AND YUET WAI KAN Howard Hughes Medical Institute Laboratorv, Division of Genetics and Molecular Hematology of the Department of Medicine, University of California-San Francisco, San Francisco, California 94143 Received 27 December 1983/Accepted 2 May 1984
We studied the effects of gene amplification on human globin gene expression in Chinese hamster ovary cells. The normal human a-globin gene (Na2) and a hybrid gene (MaG) containing the 5' promoter-regulator region of the mouse metallothionein gene and the human structural aL2-globin gene were linked to a modular SV2cDNA dihydrofolate reductase (DHFR) gene. The recombinant DNA molecules were introduced into Chinese hamster ovary cells by calcium phosphate precipitation. After initial selection to retain the DHFR and linked sequences, the cells were cultured in increasing concentrations of methotrexate up to 0.2 mM. Southern blot analysis of total cellular DNA showed an approximately 500- to 1,000-fold increase in the number of copies of DHFR and human ot-globin genes. The transcription of the a-globin and DHFR genes increased as their copy number within the cells increased. The transcription of the amplified hybrid MaG gene was also inducible with cadmium treatments. Both mature mRNA and "read-through" transcripts were observed. DHFR constituted approximately 10% of pulse-labeled cellular proteins in these cells, but no human a-globin was detected. In vitro translation of polyadenylated RNA from these cells showed that a-globin mRNA transcribed from the amplified a-globin genes was functional and directed a-globin chain synthesis. In situ hybridization of 3Hlabeled oa-globin and DHFR DNA probes in chromosome preparations from the two cell lines indicated that both genes were coamplified in the same chromosomal locations in each cell type. These results indicate that gene amplification enhances human globin gene expression in cultured Chinese hamster ovary cells.
some-like structures called double minutes (6, 20). Subsequent studies showed that cloned DHFR genes and other genes cotransformed with them could also be amplified by this stepwise MTX selection when they were transferred into DHFR-deficient cells (10, 21, 22, 32, 35). In these experiments, we linked a normal human a2-globin gene (Nac2) and the MaG hybrid gene to a cloned modular DHFR gene and transferred and amplified these sequences in Chinese hamster ovary (CHO-Kl) cells deficient in DHFR. The a-globin was coamplified with the DHFR genes and was efficiently transcribed and stably integrated into the CHO-Ki chromosomes. MATERIALS AND METHODS Construction of transforming plasmids. Construction of vectors and their recombinants was performed as described previously (27). Restriction enzymes were purchased from Boehringer Mannheim Biochemicals and Bethesda Research Laboratories. The plasmid pSV2-DHFR (39) was first digested with restriction enzymes PvuII and BamHI and size fractionated on agarose gel. The 1.9-kilobase pair (kbp) DNA fragments containing the intact modular SV2-DHFR gene and the simian virus 40 (SV40) origin of replication and enhancer sequences were isolated by glass bead extraction (44). Polynucleotide linkers (Collaborative Research, Inc.) containing the BamHI recognition sequences were ligated to the PvII ends. The resulting BamHI fragments were then inserted into the BglII site of a modified pBR322 plasmid which was derived from the cosmid pHC79 (18) by deleting the cohesive end segment between two BglII sites. This recombinant plasmid vector was designated pMV104. The Na2-globin gene in the 1.5-kbp PstI fragment was obtained from plasmid SSt2 (26). The hybrid MaG-globin gene was derived from plasmid pMa2 (Y.-F. Lau. unpublished data) by digestion with EcoRI and BamHI. The respective gene fragments were purified by agarose gel electrophoresis and
DNA-mediated gene transfer to mammalian cells has provided a means of studying the function and structure of cloned eucaryotic genes and has raised the possibility of gene replacement in genetic disorders such as sickle cell anemia and thalassemia. In previous studies, globin genes introduced stably in heterologous cells were not expressed at a high level (25, 28, 45). In an effort to increase the expression of transfected globin genes, we used mammalian inducible promoters such as the mouse metallothionein (MTI) gene to enhance human globin gene transcription. In an experiment involving a hybrid gene (MaG) that contains the 5' promoter-regulator sequence of the mouse MT-I gene and the coding sequences of the human a-globin gene, we showed that the transcription of this hybrid gene could be regulated with inducer molecules such as cadmium (12, 14, 31). However, the amount of mRNA produced was still insufficient to support adequate globin translation (Y.-F. Lau et al., manuscript in preparation). In this study, we explored gene amplification as a means of enhancing human globin gene expression. The amplification of the dihydrofolate reductase (DHFR) genes in cells resistant to the chemotherapeutic agent methotrexate (MTX) has been well documented (1, 36). Cells grown in increasing concentrations of MTX developed resistance to the drug by overproducing its target enzyme, DHFR, as a result of amplification of the endogenous DHFR genes within the genome. In sta,ble transformants, the amplified DNA sequences usually reside in homogeneous staining regions of the chromosomes (11, 34), whereas unstable transformants harbor the DHFR sequences in small chromo* Corresponding author. t Present address: Divisions of Pediatrics and Medical Biochemistry, The University of Calgary, Calgary, Alberta, Canada T2N 4N1. 1469
1470
LAU. LIN. AND KAN
glass bead extraction and were subcloned into the corresponding sites in pMV104. Cell culture, transformation, and MTX selection. CHO-Kl cells deficient in DHFR (42) were maintained in Dulbecco modified Eagle medium (GIBCO Laboratories) supplemented with penicillin-streptomycin, 10% fetal calf serum, 20 ,ug of proline per ml, and 150 p.g of glutamine per ml. Recombinant SV2-DHFR plasmids containing either Nox2- or M(xGglobin genes were transferred to the CHO-Kl cells by calcium phosphate coprecipitation (13). A 10-pLg sample of DNA-phosphate precipitates was incubated overnight with 2 x 106 CHO-Kl cells. The cells were then treated with 15% glycerol in phosphate-buffered saline for 2 min, washed with fresh phosphate-buffered saline, and cultured in complete medium for 24 h before selection in Dulbecco modified Eagle medium lacking glycine, thymidine, and purine as described previously (42). The medium was changed every 3 to 4 days for 21 days. Surviving cell colonies were pooled, grown to mass culture, and cultured in selective medium containing 0.05 F.M MTX. The cells resistant to this concentration of MTX were again grown to mass culture and further selected with higher doses of MTX. The cells underwent stepwise selections of 0.05, 0.1, 0.2, 0.4, 1, 2, 5, 10, 20, 50, 100, and 200 F.M MTX over a 3-month period. Gene amplification, transcription, and cadmium induction in transformed cells. The ot-globin and DHFR gene copy number in transformed CHO-Kl cells at different intervals of MTX selection was determined by Southern hybridization (37). After digestion with appropriate restriction enzymes, the DNA was size fractionated on 0.8% agarose gel and transferred to nitrocellulose filter paper (BA 85; Schleicher & Schuell, Inc.) as described previously (19). For transcription studies of the amplified genes, polyadenylated [poly(A) ] RNA was isolated from different cell cultures by direct binding to oligodeoxythymidylate-cellulose as described previously (43). For cadmium induction, cells were treated with either sublethal (0.2 p.M) or lethal (16 ,uM) doses of cadmium sulfate in selective medium for 10 to 12 h before RNA extraction. The amounts of specific RNA were determined by Northern hybridization as described previously (25, 41). The 1.5-kbp PstI oL-globin and the 1.9-kbp BamHI SV2-DHFR gene fragments were labeled by nick translation and used as probes in both Southern and Northern analyses. Protein synthesis. Cellular proteins were labeled in vivo with [355]methionine (Amersham Corp.) for 2 h at 37°C. The cells were washed twice with phosphate-buffered saline and scraped off the plates with a rubber stopper. They were then lysed by freeze-thawing three times. In vitro translation of poly(A)+ RNA was performed with rabbit reticulocyte lysate and wheat germ extracts (Bethesda Research Laboratories). Immunoprecipitation of translated products with an antibody against human x-globin (a gift from S. Boyer) was performed as described previously (40). All protein analyses were performed in 15% sodium dodecyl sulfate (SDS)polyacrylamide gels (24). In situ chromosome localization. In situ hybridizations of 3H-labeled ao-globin or DHFR DNA probes to chromosome preparations were performed as described previously (15). Both gene probes were nick translated to a specific activity of 2 x 107 cpm/,ug with [3H]dATP, [3-I]dCTP, and [3HJTTP (Amersham). Hybridization of these DNA probes to chromosome preparations was performed in a hybridization mixture containing 50% formamide and 10% dextran sulfate at 37°C for 16 to 20 h. Autoradiography was performed with Kodak NTB2 emulsion. Exposure time varied from 2 to 7 days.
MOL. CELL. BIOL.
RESULTS Plasmid construction. The modular DHFR gene from plasmid pSV2-DHFR was inserted into a modified p3R322 derived from cosmid pHC79. This DHFR gene-containing plasmid, designated pMV104, was then used as a vector for cloning the human ot-globin gene. Two different ot-globin genes were inserted into this vector: a 2.75-kbp Ec oRIBamHI hybrid mouse metallothionein/human co-globin gene (McxG) in which the human ot2-globin gene is under the control of the metallothionein promoter, and a 1.5-kbp PstI fragment derived entirely from the human ot2-globin gene. The maps of these two plasmids are shown in Fig. 1. Transformation and amplification of DHFR and linked oaglobin sequences. The transformation efficiencies of the plasmids to the CHO-Kl cells deficient in DHFR ranged from two to four colonies per 104 cells. After selection to retain the DHFR and linked sequences, the transformed CHO-Kl cells were pooled and grown in medium containing 0.05 p.M MTX. MTX concentration was increased stepwise to 0.2 mM over a 3-month period. To monitor the amplification process of the transforming sequences, DNA was isolated from the CHO-KI cells at different MTX concentrations and analyzed by Southern hybridization (Fig. 2). Both DHFR and linked globin DNA sequences were amplified in MTX-resistant cells. The transformed CHO-Ki cells initially contained approximately one copy per cell in MoxG-transformed cells, and approximately two copies per cell in Nox2transformed cells. As the transformed cells became resistant to 0.1 and 0.2 mM MTX, the intensity of the ax-globinspecific bands increased markedly, representing ca. 500 to 1,000 copies of the ox-globin genes in each cell line. After digesting DNA from cells containing the amplified MotG genes with E coR1 and BamHI to release the inserted globin genes from the plasmid, we observed two major cxglobin DNA species, one corresponding to the original 2.75kbp input sequence and the other representing a small, 1.4kbp ot-globin fragment. Additional large-molecule species were observed when a large amount of DNA was loaded onto the gel. After DNA from these cells was digested with EcoRI which cut the input plasmid once, the MaG genes BgIII/BamrHl
a. MT-I
-
_
MaG Eco RI
b.
a2/
a Globin 7
,'Bam HI
PstI
Pst
pMV 104
BglII/
BamHI AvaI SV40 on Bam Hi/ Hind II Bgg Blll
BamHl
,cR
pMV 104
BgIII/
AvaI
BamHIl SV40o
Bam HI!
DHFR
Hind II
Bglll
BgIII
FIG. 1. Construction of recombinant DNA plasmids containing human a-globin genes and the modular SV2-DHFR selectable marker. (a) The hybrid globin gene MaG was inserted in the EcoRIBamHI site of the plasmid vector pMV104. The stippled section of the insert represents the metallothionein portion of the hybrid gcne. (b) The 1.5-kbp PstI fragment harboring the intact normal a2-globin gene was inserted in the PstI site of pMV104. The lightly dotted boxes represent SV40 sequences within the modular SV2-DHFR gene. The darker boxes represent approximately 0.5 kbp of bacteriophage sequences present in the original pHC79 cosmid.
VOL. 4. 1984
EXPRESSION OF AMPLIFIED HUMAN ox-GLOBIN GENES
ax-Globin probe
1471
DHFR probe r~~~~~~~~~~~~~~~~~~ ~~~~~I
Human CHO
MaG
Pst I
Na2 EcoRI
Bam HI/Eco Rl
I
Pst I
I~~~~~~~~~~~~~~~~~~~~~~~~~~~~
I~~~~~~~~~~~~~~~~~
McxG
1
Hind IIl
Eco RI "I
*
Na2
Ir
q *$
16.5
)
14
