Gene amplification in mammalian cells: strategies for protein production Rodney E. Kellems Baylor College of Medicine, Houston, Texas, USA
Gene amplification is an experimental strategy for increasing protein production in mammalian cells. Co-amplification of the target gene by genetically linking it to one or more selectable and amplifiable genetic markers is a particularly successful strategy. A number of papers published in the past year or two illustrate the use of gene amplification to achieve high levels of expression.
Current Opinion in Biotechnology 1991, 2:723-729
Introduction
Table 1. Drug resistanceand gene amplificationin mammalian cells.
A common strategy for producing large quantities of desired proteins in mammalian cells involves covalent linkage of a transcription unit encoding a protein of interest to an amplifiable marker followed by cotransfer and coamplification in an appropriate recipient cell [1°°]. A large number of amplifiable markers are now available to the genetic engineer for use in protein overproduction in mammalian cells (Table 1). Some amplifiable markers are designed primarily for use with cells having specific genetic deficiencies, whereas others can be used with a broad range of mammalian cell types. The selection schemes that have resulted in the highest levels of amplification are those involving the use of specific inhibitors of essential enzymes. Selective conditions should be specific and yield only cells with the desired phenotype. If a high level of amplification is desired it is important that the amplification process is not limited by cellular toxicity resulting from the product of the amplifiable marker gene or the drugs used in the selection process. It is preferable that the selective agents required for the amplification protocol are inexpensive, stable, and readily available. As we shall see below, it is desirable that an amplification scheme is compatible with other amplification strategies thereby allowing the independent amplification of multiple genes. Two amplifiable markers that meet most of these criteria are dihydrofolate reductase (DHFR) and adenosine deaminase (ADA). In this review I will describe examples from the recent literature that illustrate a number of circumstances in which these markers have been used alone and in combination to achieve high levels of protein production in mammalian cells.
Selectiveagent
Gene amplified
Methotrexate Dihydrofolate reductase Metallothionein Cadmium N-(phosphonacetyl)-L-aspartate CAD Adenosine deaminase 2'-Deocycoformycin AMP deaminase Coformycin 6-Azauridine, pyrazofurin UMP synthetase IMP 5'-dehydrogenase Mycophenolic acid 5-Fluorodeoxyuridine Thymidylate synthetase P-glycoprotein 170 Multiple drugs Ribonucleotide reductase Aphidicolin or hydroxyurea ~-Aspartyl hydroxamate,albizziin Asparagine synthetase Ornithine decarboxylase ~-Difluoromethylornithine HMG-CoA reductase Compactin N-acetylglucosaminyltransferase Tunicamycin Threonyl-tRNA synthetase Borrelidin Histidine-tRNA synthetase Histidinol Na+-K +-ATPase Ouabain This table is adapted from reference[1"el which should be consulted for most of the original citations. See references[26-29] for additional relevant citations.
Dihydrofolate reductase: the original amplifiable marker The most widely employed gene transfer and amplification strategy involves the use of DHFR expression vectors in conjunction with DHFR-deficient Chinese hamster ovary (CHO) cells. CHO cells are well suited to protein
Abbreviations ADA--adenosine deaminase; CAD--carbamoyl phosphate synthetase, aspartate transcarbam0ylase and dihydroorotase; CHO--Chinese hamster ovary; dCF~2'-deoxycoformycin; DHFR~ihydrofolate reductase; GRP--glucose regulated protein; hGH--human growth hormone; MTX--methotrexate; PALA--N-(phosphonacetyl)-t-aspartate; t-PA--tissue-type plasminogen activator; vWF--von Willebrand factor.
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Expressionsystems production and for this reason have been used to produce a number of commercially valuable proteins [1.°]. Expression vectors encoding proteins of interest as well as DHFR are introduced into recipient cells, DHFR positive transformants are isolated and populations of transformed cells selected for increasing levels of resistance to methotrexate (MTX), an inhibitor of DHFR. As a result of this procedure it is possible to isolate MTX-resistant cells that have increased levels of DHFR protein and amplified copies of DHFR genes. The gene of interest is coamplified with DHFR because of its close physical proximity. This approach to protein overproduction has been highly successful and remains the most frequently used cotransfer and coamplification strategy.
Adenosine deaminase: a dominant amplifiable genetic marker Unlike DHFR, ADA is not normally required for cell growth in culture, but culture conditions have been devised in which cell growth depends on ADA activity [2,3]. Under these conditions it is possible to use a potent and specific inhibitor of ADA activity, 2'-deoxycoformycin (dCF), to select for cells with increased levels of ADA activity. Because of the relatively low levels of endogenous ADA found in most mammalian cells, it is relatively easy to isolate transformants because of their increased ADA expression provided by the input ADA mini-gene. For this reason ADA can function as a dominant amplifiable genetic marker in mammalian cells [4]. Extremely high levels of ADA are not toxic to cells, as indicated by the existence of cells with highly amplified ADA genes in which the encoded enzyme accounts for 75% of the soluble protein [5]. ADA selection protocols are compatible with other amplification schemes, such as MTX selection for amplified DHFR genes [6]. As shown below, this feature allows for the simultaneous, alternating, or consecutive use of ADA and DHFR selection protocols to accomplish a number of objectives conceming protein production in mammalian cells.
Two amplifiable markers are better than one High level synthesis of multi-suhunit proteins The use of genetic engineering to construct recombinant monoclonal antibodies with novel functions is having a major impact on many areas of biotechnology and medicine. The process of antibody engineering is being used to create 'humanized' hybrid antibodies in which the heavy chain constant region of murine monoclonals is replaced with human constant regions. Similarly, constant regions may be replaced with toxins to create immunotoxins with cell-type specificity. Additional possibilities include the use of protein domains encoding enzymatic activities that improve the convenience and sensitivity of enzyme-linked immunosorbent assays. Large quantities of recombinant antibodies are required for these therapeutic and diagnostic purposes. The use of gene amplification strategies to independently cotransfer and coamplify genes encoding recombinant heavy and light
chains provides an important strategy for antibody production in mammalian cells. This strategy is well illustrated by a recent publication by scientists at Genetics Institute. Wood et al. [7"] isolated cDNAs encoding heavy and light chains capable of assembling to form antibodies that specifically recognize the hapten 4-hydroxy-3-nitrophenacetyl. Light and heavy chain cDNA transcription units were linked to DHFR and ADA markers, respectively, and introduced independently into different populations of CHO cells. Pools of transformants expressing either light or heavy chains were independently isolated and selected for resistance to increasing concentrations of MTX or dCF, respectively. Cells expressing both heavy and light chains were obtained by cell hybridization and the resulting hybrids were selected for yet higher levels of resistance to both MTX and dCF. Media from cells expressing heavy or light chain had no 4-hydroxy-3-nitrophenacetyl binding activity, whereas media from hybrid cells contained IgM that exhibited specific hapten binding and complement fixation, and closely resembled the original hybridoma antibody. Cells obtained in this way produced antibody at a rate of approximately 67 I.tg/106 cells/48 hr. According to Wood et al. the ability to select independently for increases in expression of light and heavy chains is advantageous for optimizing the production of functional antibody by the CHO cells.
Genetic modification of the secretory pathway Glucose regulated proteins (GRPs) belong to the class of stress-induced proteins that includes the heat shock proteins. The GRPs are found predominantly in the endoplasmic reticulum as 78 and 94 kD proteins referred to as GRP78 and GRP94. The GRPs have considerable sequence similarity to the heat shock proteins that are found predominantly in the cytoplasm as 70 and 90 kD proteins. The immunoglobulin heavy chain binding protein termed BiP is identical to GRP78; BiP associates with free immunoglobulin heavy chains in the endoplasmic reticulum, and prevents their secretion until they have assembled with light chains. It is believed that GRP78 also associates with improperly folded or underglycosylated proteins and prevents their secretion. Domer et al. [8] have found that secretion of a genetically modified form of tissue-type plasminogen activator (t-PA) that lacks potential glycosylation sites is considerably reduced by its strong association with GRP78. This association is believed to block the transport of the recombinant t-PA from the endoplasmic reticulum to the Golgi apparatus. Domer et al. found that the introduction and amplification of GRP78 antisense constructs resulted in a threefold reduction in GRP78 levels and a corresponding increase in secretion of the recombinant t-PA. Additional studies indicated that in these cells t-PA showed a reduced association with GRP78, and that a greater portion was processed to the mature form and secreted. Two amplifiable markers were used to accomplish the goals of these experiments. DHFR was used as the amplitiable marker to prepare cells producing high levels
Gene amplification in mammalian cells: strategies for protein production Kellems of the recombinant t-PA. Following this, ADA was used as the amplifiable marker to selectively isolate cells with amplified copies of the vector expressing antisense RNA against GRP78 mRNA. In some cases the latter approach resulted in the isolation of cells showing a 10-fold reduction in GRP78 expression. These results illustrate that in cases where an association with GRP78 in the endoplasmic reticulum is inhibitory to secretion, the reduction of GRP78 synthesis by the use of amplified copies of antisense constructs can result in increased secretion of the desired product.
Coexpression of cooperating proteins Factor VIII is an essential participant in the intrinsic pathway of blood coagulation. Hemophilia A is an X-linked bleeding disorder resulting from Factor VIII deficiency. Treatment for this condition has traditionally involved administration of a Factor VIII concentrate derived from human plasma. Although this treatment has merit, serious problems remain, including cost, limited availability, and the significant possibility of life-threatening viral contamination. A major advance occurred with the cloning of the
Factor VIII cDNA. Initial efforts to overproduce Factor VIII cDNA in CHO cells by coamplification with DHFR met with difficulty [9]. The amount of Factor VIII recovered from the media of cells having a 500-fold coamplification of the Factor VIII gene was approximately 2-3 orders of magnitude below that observed when genes encoding other proteins [e.g. erythropoietin, yon Willebrand factor (vWF)] were comparably amplified. One reason fbr the reduced recovery of Factor VIII is the requirement for vWF in the culture medium to promote stable assembly and to provide proteolytic protection of the secreted Factor VIII. Kaufman et al. [10] assessed the requirement for the vWF by deriving cell lines in which Factor VIII and vWF were coamplified. They began with cells in which a Factor VIII expression vector was amplified after linking it with DHFR (Fig. 1). A vWF expression vector linked to ADA was introduced into these cells. Transformants were isolated and coamplification of vWF via linkage to ADA was initiated. At each step in the amplification process the production of vWF and Factor VIII was determined. The results demonstrated that vWF production increases over 200-fold with increasing ADA selection. As
Introduce into DHFR CliO Cells
ilciiV r cDNA
Select for resistance to MTX
DHFR cDNA
@@ MTX resistant cells produce Factor VIII
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Introduce into Factor \VIII producing I cells
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I resisl F .............
yon Witlebrand factor
Fig. 1. Coexpression of von Willebrand factor (vWF) and Factor Viii eliminates the requirement for exogenously added vWF and increases the yield of Factor VIII. Introduce the vector, pRxPy VIllI and select transfected (MTX-resistant) cells. This produces a low yield of unstable Factor VIII. Cells that co-express both Factor VIII and vWF were obtained by initial transfer and amplification of the Factor VIII expression vector and subsequent transfer and amplification of the vWF expression vector [10]. As the vWF expression vector was amplified over a 200-fold range there was a corresponding increase in the level of Factor VIII activity in serum-free medium.
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Expressionsystems the level of vWF expression increased there was a corresponding increase in the recovery of Factor VIII from serum-free culture medium. The high level of vWF expression achieved through coamplification with ADA replaced the requirement for serum as a source ofvWF and substantially improved the recovery of Factor VIII from genetically engineered mammalian cells.
Highly inducible expression vectors In some cases it will be desirable for a gene of interest to remain inactive during the process of cotransfer and coamplification. This may be necessary because excessive quantities of the gene product may retard cell growth. In other cases the investigator may wish to keep the gene inactive until it is desirable to activate its expression. The latter could be timed to meet specific experimental demands or production requirements. Israel and Kaufman [11] used ADA selection protocols to introduce and amplify a mini-gene encoding the rat glucocorticoid receptor into DHFR-deficient CHO cells. The resulting cells, termed GRA, produce receptor protein at 5-10 times the level found in rat liver or hepatoma cell lines. Because GRA cells are DHFR deficient, they can be used as a host for DHFR based amplification of heterologous genes placed under control of glucocorticoid regulatory elements. In one example, Israel and Kaufman used a bicistronic expression vector containing a glucocorticoid-inducible transcription unit encoding t-PA and a 3' open reading frame encoding DHFR. The vector was introduced into GRA cells and amplified by selection for MTX resistance. At all levels of MTX selection the presence for glucocorticoid resulted in a 5-10 fold increase in t-PA synthesis. Thus, stable inducible expression of amplified genes at relatively high levels can be achieved with this approach.
Double your chances of success The desired gene may not remain faithfully coamplifed with the amplifiable marker as a result of DNA rearrangements and recombination that are known to occur during the amplification process. Because of this it is necessary periodically to isolate and analyze subclones of the selected populations in order to identify those that have faithfully coamplified the nonselectable gene of interest. To reduce the time and effort required for this analysis, Kellems et al. constructed a double selection vector in which a gene of interest, in this case the t-PA gene, is flanked by two amplifiable markers, ADA and DHFR [6]. This plasmid was introduced into DHFR-deficient CHO cells and transformants selected for increased ADA expression. Because ADA is a dominant selectable marker, it is preferable to utilize ADA selection to isolate the initial transformants. These were expanded and altemately selected for increased ADA and DHFR. The level of t-PA expression was monitored as a function of increased selection pressure. The results indicate that a linked gene is more faithfully coamplifed when sandwiched between two amplifiable markers. These findings suggest that the placement of a nonselectable gene between ADA and DHFR may serve as a generally useful strategy for the co-
transfer and coamplification of genes that encode commercially valuable proteins.
Increasing the odds Gene amplification occurs at discrete loci in mammalian cells at frequencies as high as 10-4. The mechanism is not understood but significant efforts are underway to determine the initial events of this process [12,13",14-]. Although the mechanism is not yet understood, genetic and environmental factors have been identified that increase the frequency of gene amplification. Some of these findings present useful techniques for the genetic engineer who is interested in constructing mammalian cells with amplified copies of specific genes.
Genetic elements that increase the frequency of gene amplification Two research groups have identified cis-acting elements that increase the frequency of gene amplification. Stanners and his colleagues [15-.-17"*] have described a middle repetitive element, termed HSAG, that acts in cB to promote amplification of DHFR mini-genes when introduced into a variety of mammalian cell lines. The presence of this element on a DHFR expression plasmid reproducibly increased the production of colonies resistant to 2 btM MTX by 20-200 fold. The DHFR genes in the resulting colonies were amplified 10-20 fold. The dement also enabled the transferred mini-genes to be amplified at a faster ,'ate. The amplification activity was localized to a 1.45 kb fragment that contains Muqike elements, inverted repeats, and A/T-rich regions. Grummt and colleagues [18"] have identified a 370bp element from the non-transcribed spacer region of murine ribosomal RNA genes that increases the copy number of a selectable marker when introduced into recipient cells. The effect on copy number was localized to an A/Trich region at the 5' end of the element and an 11 bp palindrome at its 3' end. Each region interacts with specific cellular proteins. Specific point mutations within the 11 bp palindrome reduced protein/DNA interactions and weakened its ability to stimulate gene amplification. The 11 bp palindrome is conserved in the otherwise strongly divergent, non-transcribed spacer regions of mouse, rat and man, and is also found in the origin/enhancer region of the human papovavirus JC. The mechanisms by which these elements function to promote amplification are not known. McArthur and Stanners [15"] favor a mechanism whereby their element promotes amplification through recombinational mechanisms. Grummt and colleagues [18 "°] favor a replicational mechanism for the element they discovered. Regardless of the mechanism, the existence of cB-regulatory elements that stimulate amplification of linked genes suggests potentially useful applications. Such dements could be included in gene amplification vectors to increase the number of tranformants initially obtained and the rate at which the associated genes are subsequently amplified.
Gene amplification in mammalian cells: strategies for protein production Kellems 727 This could result in saving considerable time and material. Cell lines in which amplification occurs at an unworkably low frequency may become amenable to gene transfer and amplification with the help of these elements.
Cell lines with amplifier phenotypes Gene amplification is a very rare event in normal diploid human and rodent cells [19,20] but it is readily detected in tumors and in permanent cell lines. These findings suggest that genetic changes associated with cellular transformation contribute to the occurrence of gene amplification. Based upon these findings Guilotto, Stark and their colleagues [21] have taken a genetic approach to identify biochemical defects that contribute to the gene amplification process. To do this they selected baby hamster kidney cells with resistance to MTX and N-(phosphonoacetyl)-L-aspartate (PALA) - - an inhibitor of carboamoyl phosphate synthetase, aspartate transcarbamoylase and dihydroorotase (CAD), a multifunctional enzyme catalyzing the first three steps of pyrimidlne metabolism - - and showed that resistance was due to the simultaneous amplification of DHFR and CAD genes. Subsequent analysis showed that the cells that are resistant to MTX and PALA(termed MP cells), showed an increase of as much as 25-fold in the frequency of resistance to coformycin, pyrazofurin, or ouabain (see Table 1 for the enzymes inhibited by these drugs). The pyrozofurin-resistant cells were analyzed and shown to have amplified copies of the UMP synthetase gene. Accordingly, resistance to coformycin and ouabain was presumed to result from amplification of genes encoding AMP deaminase and Na +,K + -ATPase, respectively. The MP cells retained the amplifier phenotype even when resistance to MTX and PALA was lost following extended growth under non-selective conditions. These findings suggested that the MP cells have a heritable change in one or more genes involved in the gene amplification process. Recent phenotypic and genetic analysis of the MP cells revealed that they have a deficiency in the repair of ultraviolet and mitomycin C induced DNA damage [22..]. The amplifier cell yielding the greatest increase in the frequency of gene amplification showed no significant alteration in cell cycle timing. Such cell lines may offer advantages for gene transfer and amplification strategies by reducing the time required to obtain cell lines with amplified copies of desired genes.
Environmental factors affecting gene amplification A number of mutagenic treatments are known to increase the frequency of gene amplification by as much as 10-100 fold [1°°]. In most cases the increase in gene amplification frequency is more than offset by the percentage of cells killed as a result of the mutagenic treatment. Although information regarding the effects of specific mutagenic treatments on gene amplification may prove usefill in understanding the mechanism of this process, such treatments are not routinely used to improve gene transfer and amplification strategies for protein production.
Gene amplification and retrovirus vectors Better titers through amplification Considerable attention is currently devoted to the development of gene therapy protocols for the treatment of certain human genetic disorders. Somatic gene therapy for Factor VIII could entail the introduction of a functional Factor VIII gene into self-renewing hematopoietic stem cells, followed by continual expression of the introduced gene in specific blood cell progeny. Successful accomplishment of this goal would provide a constant and self-renewing source of recombinant Factor VIII and eliminate the need for repeated transfusions of the recombinant protein. Retrovirus vectors are considered to be the most efficient means for introduction of genetic information into recipient cells. The efficiency of gene transfer is often limited by the titer of the replication defective retrovirus vectors utilized in this process. Gene amplification can be very useful to increase viral citer. Following an initial lead provided by Miller et al. [23], who used DHFR amplification to improve viral titers, Israel and Kaufman [24--] have recently reported using coamplification with ADA to improve the citer of replication defective retroviruses capable of transducing Factor VIII. To do this they constructed a retroviral vector encoding Factor VIII and ADA. This construct was introduced into a retroviral packaging cell line and transformants were selected for increased ADA expression. The resulting transformants secreted Factor VIII into the medium and produced replication defective retroviruses capable of transferring Factor VIII and ADA expression to recipient cells. Selection of viral producer cell lines for increased ADA expression yielded cells with a 20-fold increase in both Factor VIII expression and viral citer. These results demonstrate the feasibility of retroviral vector mediated transfer of Factor VIII expression and also the utility of amplifiable markers for increasing viral titer.
Ping-pong amplification without selection Kozak and Kabat [25 ,°] have developed a procedure termed 'ping-pong amplification' by which retrovirus vectors are used to achieve high levels of protein production in mammalian cells. They found that recombinant retroviral vector DNA encoding the human growth hormone (hGH) gene was rapidly amplified when added to cultures containing a mixture of ecotropic (Psi-2) and amphotropic (PAl2 or PA317) packaging cell lines. Under these coculture conditions helper-free virions released from one cell type infect the other, and the result is a theoretically limitless back-and-forth (ping-pong) process of vector transduction between the two cell types. Kozak and Kabat demonstrated that ping-pong vector amplification leads to a high level of hGH production. The amplification process is terminated by isolating individual clones of ecotropic or amphotropic packaging cell lines from the coculture. Stable clones were obtained that produce hGH at levels amounting to 4-6% of the secreted protein. The authors indicate that this method has been used to produce human erythropoietin, the erythropoietin receptor, the erythroblast mitogenic glycoprotein encoded by Friend erythroleukemia virus, the mouse ectopic retro-
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Expressionsystems vires receptor, and a brain protein. The method, therefore, appears to have significant potential for protein production in mammalian cells.
Conclusion T h e ability t o i n t r o d u c e n e w g e n e t i c i n f o r m a t i o n i n t o m a m m a l i a n cells a n d s e l e c t t r a n s f o r m a n t s w i t h desired p r o p e r t i e s offers e n o r m o u s c o m m e r c i a l p o t e n t i a l . T h e ability t o s e l e c t cells t h a t h a v e a m p l i f i e d c o p i e s o f t h e introduced genes adds considerably to the commercial potential o f this p r o c e s s . O n e o f t h e m o s t s u c c e s s f u l strategies f o r o b t a i n i n g a h i g h level o f e x p r e s s i o n o f h e t e r o l o g o u s g e n e s i n m a m m a l i a n cells h a s b e e n b y coamplification of desired genes by genetic linkage with selectable a n d a m p l i f i a b l e m a r k e r s . T h e availability o f a c o l l e c t i o n o f a m p l i f i a b l e g e n e t i c m a r k e r s will p l a y a n i n c r e a s i n g l y i m p o r t a n t r o l e i n a l l o w i n g g e n e t i c e n g i n e e r s to m a n i p u late m a m m a l i a n cells as t h e y w i s h . T h e ability to e n g i n e e r m a m m a l i a n cells p r o d u c i n g l a r g e q u a n t i t i e s o f d e s i r e d RNA a n d p r o t e i n p r o d u c t s h a s o p e n e d u p n e w possibilities o f c o n s i d e r a b l e i m p o r t a n c e t o b i o l o g y a n d m e d i c i n e .
References and recommended reading Papers of special interest, published within the annual period of review, have been highlighted as: • of interest •. of outstanding interest KAUFMANRJ: Selection and Coamplification of Heterologous Genes in Mammalian Cells. Meth Enzymol 1990, 185:537-566. This is one of several very useful reviews dealing with expression of heterologous genes in mammalian cells that appear in this volume of Methods in Enzymology. This article reviews the methods, genetic mark ers, and strategic considerations for using gene amplification to achieve high levels of protein production in mammalian cells. It is a thorough and accurate account of over 10 years of research in this area. 1.
•.
2.
YEUNG C-Y, INGOLtA DE, BOBONIS C, DUNBAR BS, RISER ME, SIC1LIANOMJ, KELLEMSRE: Selective Overproduction of AdenosIne Deaminase in Cultured Mouse Cells. J Biol Chem 1983, 258:8338-8345.
3.
YEUNGC-Y, FRAYNE EG, AL-UBAIDI MR, HOOK AG, INGOLIA DE, BOBONIS C, WRIGHT DR, KELLEMSRE: Amplification and Molecular Cloning of Murine Adenosine Deaminase Gene Sequences. J Biol Chem 1983, 258:15179-15185.
4.
KAUFMANRJ, MURTHAP, INGOIaADE, YEUNG C-Y, KELLEMSRE: Selection and Amplification of Heterologous Genes Encoding Adenosine Deaminase in Mammalian Cells. Proc Natl Acad Sci USA 1986, 83:3136-3140.
5.
INGOLIADE, YEUNG C-Y, ORENGO IF, HARRISON ML, FRAYNE EG, RUDOLPHFB, KELLEMSRE: Purification and Characterization of Adenosine Deaminase from a Genetically Enriched Mouse Cell Line. J Biol Chem 1985, 260:13261-13267.
6.
KELLEMSRE, JOHNSTON EM, WARD KE, LITTLE SP: Adenosine Deaminase: a Dominant Amplifiable Genetic Marker for use in Mamnlalian Cells. In Genetics a n d Molecular Biology of Industrial Microorganisms edited by Hershberger CL, Queener SW, Hegeman G [book]. Washington: American Society for Microbiology 1989, pp 215-225.
7. •.
WOOD CR~ DORNER AJ, MORRIS GE, ALDERMANEM, WILSON D, O'HARA RM, KAUFMANRJ: High Level Synthesis of hnmunoglobulins in Chinese Hamster Ovary Cells. J I m m u n o l 1990, 145:3011-3016. The work presented here shows that a functional antibody can be produced in genetically engineered CHO cells at levels equivalent to or greater than many hybridomas. This was achieved by transfecting different CHO ceils with light and heavy chain expression vectors linked to DHFR and ADA, respectively, for use as an amplifiable marker. Following amplification in independent ceil lines, cell hybrids were obtained that produced functional antibody at a high rate. The expression strategy employed here should prove useful for the production of genetically engineered antibodies and other multi-subunit proteins. 8.
DORNERAJ, KRANE MG, KAUFMANRJ: Reduction of Endogenous GRP78 Levels Improves Secretion of a Heterologous Protein in CHO cells. Mol Cell Biol 1988, 8:4063-4070.
9.
KAUFMANRJ: Genetic Engineering of Factor VIII. Nature 1989, 342:207-208.
10.
KAUFMANRJ, WASLEYLC, DAVIESMV, WISE RJ, ISRAELDI, DORNER AJ: Effect of yon Wlllebrand Factor Coexpression on the Synthesis and Secretion of Factor VIII in Chinese Hamster Ovary Cells. Mol Cell Biol 1989, 9:1233-1242.
11.
ISRAELDI, KAUFMANNRJ: Highly Inducible Expression from Vectors Containing Multiple GREs in CHO Cells Overexpressing the Glucocorticoid Receptor. Nucleic Acids Res 1989, 17:4589-4604.
12.
TRASKBJ, HAMIaNJL: Early Dihydrofolate Reductase Gene Amplification Events in CHO Cells Usually Occur on the Same Chromosome Arm as the Original Locus. Genes Dev 1989, 3:1913-1925.
13. .
SMITHKA, GORMANPA, STARKMB, GROVESRP, STARKGI~ Distinctive Chromosomal Structures are Formed Very Early in the Amplification of CAD Genes in Syrian Hamster Cells. Cell 1990, 63:1219-1227. Fluorescence in situ hybridization to metaphase chromosomes was used to visualize recently amplified arrays of CAD genes. The results indicate that very large regions of DNA, each as long as 10kb, are tandemly arrayed near one of the original CAD genes that remained at its original location. The tandem arrays of amplified DNA are believed to arise via sister chromatid exchange. 14. •
W1NDLEB, DRAPER BW, YIN Y, O'GORMAN S, WAHL GM: A Central Role for Chromosome Breakage in Gene Amplification, Deletion Formation, and Amplification Integration. Genes Dev 1991, 5:160-174. Like [13"], this paper also reports fluorescence in situ hybridization to examine the early products generated during amplification of the single DHFR locus present in a line of CHO ceils. Windle et al. show that cells having undergone DHFR gene amplification are prone to deletion of the DHFR gene and flanking sequences at the original locus. These findings, in contrast to those presented in [12] and [13"], implicate chromosome breakage as an early or initial event in the amplification process. 15. .,
MCARTHURJG, STANNERS CP: A Genetic Element that Increases the Frequency of Gene Amplification. J Biol Chem 1991, 266:6000~005. In this manuscript Mcarthur and Stanners show that a mammalian repetitive sequence element, HSAG-1, can act in cis, to increase the frequency of amplification of a vector following gene transfer into mammalian cells, the s~nulation of amplification was as much as 200 fold and the effect was observed in a variety of mammalian ceils lines. The genetic element thus identified could be a useful addition to mammalian gene amplification vectors. 16. ,.
BRITEL LK, MCARTHUR JG, STANNERS CP: Sequence Requirments for the Stimulation of Gene Amplification bya Mammalian Genomic Eloment. Gene 1991, 102:149-156. Deletion analysis of the HSAG-1 element referred to in [ 15"'] revealed the presence of multiple positive acting elements that are required for the amplification effect, a highly active 1,45 kb fragment was identified that contains the necessary sequence elements to promote gene am-
Gene amplification in mammalian cells: strategies for protein production Kellems 729 plitication following transfer into mammalian cells. Additional HSAG-1 like elements were tested and found to stimulate vector amplification following introduction into mammalian cells.
lected loci. Here they associate the amplifier phenotype with an altered DNA repair capability. These, or related cell lines, may prove useful as recipients in gene transfer and amplification strategies.
17. °°
23.
MCARTHURJG, BE1TEL LK, CHAMBERLAIN J, STANNERS CP: Elements w h i c h Stimulate G e n e Amplification in Mammalian Cells: Role of Recombinogenic Sequences/Structures and Transcriptional Activities. Nucleic Acids Res 1991, 19:2477-2484. Structural and Functional analysis of the HSAG-1 regulating elements revealed that the ability to stimulate amplification is orientation dependent and that the element must be transcribed in order to exert its effect. This study provides important information that is critical for proper use of these elements as components of mammalian gene amplification vectors. 18. oo
WEGNERM, SCHWENDER S, DINKL E, GRUMMT F: Interaction of a Protein with a Palindromic Sequence from Murine rDNA Increases t h e O c c u r r e n c e of Amplification-dependent Transformation in Mouse Cells. J Biol Cbem 1990, 265:13925-13932. An l l b p palindrome is described that stimulates by more than 10-fold the rate of transformation dependent amplification of a selectable marker gene. The palindromic sequence interacts specifically with proteins from mouse extracts. Mutations that reduce protein binding also reduce the stimulation of gene amplification. The sequence elements identified by these investigators may play a useful role as a component of gene amplification vectors. 19.
WRIGHTJA, SMITH HS, WATT FM, HANCOCK MC, HUDSON DL, STARK GR: DNA Amplification is Rare in Normal H u m a n Cells. Proc Natl Acad Sci USA 1990, 87:1791-1795.
20.
TLSTYTD: Normal Diploid H u m a n and Rodent Cells Lack a Detectable Frequency of Gene Amplification. Proc Natl Acad Sci USA 1990, 87:3132-3136.
21.
22. °°
GIULOTTOE, KNIGhtS C, STARK GR: Hamster Cells with Increased Rates of DNA Amplification, a N e w Phenotype. Cell 1987, 48:837-845.
GIULOTTOE, BERTONI L, ATTOLINI C, RANIALD1G, ANGLANAM: BHK Cell Lines with Increased Rates of Gene Amplification are Hypersensitive to Ultraviolet Light. Proc Natl Acad Sci USA 1991, 88:3484-3488. These authors had previously isolated a cell line [21] that is character ized by a 25-fold increase in the frequency of gene amplification at se-
MILLERAD, LAW M-F, VERMA IM: Generation of Helper-free Amphotropic Retroviruses that T r a n s d u c e a Dominant-acting, Methotrexate-resistant Dihydrofolate Reductase Gene. Mol Cell Biol 1985, 5:431-437.
24. ..
ISRAELDI, KAUFMANFJ: Retroviral-mediated Transfer and Amplification of a Functional H u m a n Factor VIII Gene. B/ood 1990, 75:1074-1080. Findings reported here demonstrate the feasibility of retroviral mediated transfer of human Factor VIII and the utility of gene amplification for increasing the viral titer. 25. •.
KOZAKSL, KABAT D: Ping-pong Amplification of a Retroviral Vector Achieves a High-level G e n e Expression: H u m a n Growth H o r m o n e Production. J Virol 1990, 64:3500-3508. These authors show that when retroviral vector DNA is added to cocultures that contain packaging cell lines with different envelope specificities, each cell type produces helper-free virions that infect the other. This ping-pong cycle of vector transduction leads to amplification of proviral sequences and a high level production of encoded proteins. 26.
LEHRMAN MA, ZHU X, KHOUNLO S: Amplification and Molecular Cloning of t h e H a m s t e r Tunicamycin-sensitive N-Acetylglucosamine-l-Phosphate Transferase Gene. T h e Hamster and Yeast Enzymes Share a C o m m o n Peptide Sequence. J Biol Cbem 1988, 263:19796-19803.
27.
KONTISKJ, ARFIN SM: Isolation of a cDNA Clone for H u m a n Threonyl-tRNA Synthetase: Amplification of the Structural G e n e in Borrelidin-resistant Cell Lines. Mol Cell Biol 1989, 9:1832-1838.
28.
TsuI FWL, ADRULISIA, MURIALDOH, SIMINOVITCHL: Amplitication of the Gene for Histidyl-tRNA Synthetase in Histidinol Resistant Chinese Hamster Ovary Cells. Mol Cell Biol 1985, 5:2381-2388.
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PAUWPG, JOHNSON MD, MOORE P, MORGAN M, F1NEMANRM, KALKAT, ASH JF: Stable Gene Amplification and Overexpression of Sodium- and Potassium-activated ATPase in Hela Cells. Mol Cell Biol 1986, 6:1164-1171.
RE Kellems, Department of Biochemistry and Institute for Molecular Genetics, Baylor College of Medicine, Houston, Texas 77030, USA.
Gene amplification is an experimental strategy for increasing protein production in mammalian cells. Co-amplification of the target gene by genetically linking it to one or more selectable and amplifiable genetic markers is a particularly successful strategy. A number of papers published in the past year or two illustrate the use of gene amplification to achieve high levels of expression.
Current Opinion in Biotechnology 1991, 2:723-729
Introduction
Table 1. Drug resistanceand gene amplificationin mammalian cells.
A common strategy for producing large quantities of desired proteins in mammalian cells involves covalent linkage of a transcription unit encoding a protein of interest to an amplifiable marker followed by cotransfer and coamplification in an appropriate recipient cell [1°°]. A large number of amplifiable markers are now available to the genetic engineer for use in protein overproduction in mammalian cells (Table 1). Some amplifiable markers are designed primarily for use with cells having specific genetic deficiencies, whereas others can be used with a broad range of mammalian cell types. The selection schemes that have resulted in the highest levels of amplification are those involving the use of specific inhibitors of essential enzymes. Selective conditions should be specific and yield only cells with the desired phenotype. If a high level of amplification is desired it is important that the amplification process is not limited by cellular toxicity resulting from the product of the amplifiable marker gene or the drugs used in the selection process. It is preferable that the selective agents required for the amplification protocol are inexpensive, stable, and readily available. As we shall see below, it is desirable that an amplification scheme is compatible with other amplification strategies thereby allowing the independent amplification of multiple genes. Two amplifiable markers that meet most of these criteria are dihydrofolate reductase (DHFR) and adenosine deaminase (ADA). In this review I will describe examples from the recent literature that illustrate a number of circumstances in which these markers have been used alone and in combination to achieve high levels of protein production in mammalian cells.
Selectiveagent
Gene amplified
Methotrexate Dihydrofolate reductase Metallothionein Cadmium N-(phosphonacetyl)-L-aspartate CAD Adenosine deaminase 2'-Deocycoformycin AMP deaminase Coformycin 6-Azauridine, pyrazofurin UMP synthetase IMP 5'-dehydrogenase Mycophenolic acid 5-Fluorodeoxyuridine Thymidylate synthetase P-glycoprotein 170 Multiple drugs Ribonucleotide reductase Aphidicolin or hydroxyurea ~-Aspartyl hydroxamate,albizziin Asparagine synthetase Ornithine decarboxylase ~-Difluoromethylornithine HMG-CoA reductase Compactin N-acetylglucosaminyltransferase Tunicamycin Threonyl-tRNA synthetase Borrelidin Histidine-tRNA synthetase Histidinol Na+-K +-ATPase Ouabain This table is adapted from reference[1"el which should be consulted for most of the original citations. See references[26-29] for additional relevant citations.
Dihydrofolate reductase: the original amplifiable marker The most widely employed gene transfer and amplification strategy involves the use of DHFR expression vectors in conjunction with DHFR-deficient Chinese hamster ovary (CHO) cells. CHO cells are well suited to protein
Abbreviations ADA--adenosine deaminase; CAD--carbamoyl phosphate synthetase, aspartate transcarbam0ylase and dihydroorotase; CHO--Chinese hamster ovary; dCF~2'-deoxycoformycin; DHFR~ihydrofolate reductase; GRP--glucose regulated protein; hGH--human growth hormone; MTX--methotrexate; PALA--N-(phosphonacetyl)-t-aspartate; t-PA--tissue-type plasminogen activator; vWF--von Willebrand factor.
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Expressionsystems production and for this reason have been used to produce a number of commercially valuable proteins [1.°]. Expression vectors encoding proteins of interest as well as DHFR are introduced into recipient cells, DHFR positive transformants are isolated and populations of transformed cells selected for increasing levels of resistance to methotrexate (MTX), an inhibitor of DHFR. As a result of this procedure it is possible to isolate MTX-resistant cells that have increased levels of DHFR protein and amplified copies of DHFR genes. The gene of interest is coamplified with DHFR because of its close physical proximity. This approach to protein overproduction has been highly successful and remains the most frequently used cotransfer and coamplification strategy.
Adenosine deaminase: a dominant amplifiable genetic marker Unlike DHFR, ADA is not normally required for cell growth in culture, but culture conditions have been devised in which cell growth depends on ADA activity [2,3]. Under these conditions it is possible to use a potent and specific inhibitor of ADA activity, 2'-deoxycoformycin (dCF), to select for cells with increased levels of ADA activity. Because of the relatively low levels of endogenous ADA found in most mammalian cells, it is relatively easy to isolate transformants because of their increased ADA expression provided by the input ADA mini-gene. For this reason ADA can function as a dominant amplifiable genetic marker in mammalian cells [4]. Extremely high levels of ADA are not toxic to cells, as indicated by the existence of cells with highly amplified ADA genes in which the encoded enzyme accounts for 75% of the soluble protein [5]. ADA selection protocols are compatible with other amplification schemes, such as MTX selection for amplified DHFR genes [6]. As shown below, this feature allows for the simultaneous, alternating, or consecutive use of ADA and DHFR selection protocols to accomplish a number of objectives conceming protein production in mammalian cells.
Two amplifiable markers are better than one High level synthesis of multi-suhunit proteins The use of genetic engineering to construct recombinant monoclonal antibodies with novel functions is having a major impact on many areas of biotechnology and medicine. The process of antibody engineering is being used to create 'humanized' hybrid antibodies in which the heavy chain constant region of murine monoclonals is replaced with human constant regions. Similarly, constant regions may be replaced with toxins to create immunotoxins with cell-type specificity. Additional possibilities include the use of protein domains encoding enzymatic activities that improve the convenience and sensitivity of enzyme-linked immunosorbent assays. Large quantities of recombinant antibodies are required for these therapeutic and diagnostic purposes. The use of gene amplification strategies to independently cotransfer and coamplify genes encoding recombinant heavy and light
chains provides an important strategy for antibody production in mammalian cells. This strategy is well illustrated by a recent publication by scientists at Genetics Institute. Wood et al. [7"] isolated cDNAs encoding heavy and light chains capable of assembling to form antibodies that specifically recognize the hapten 4-hydroxy-3-nitrophenacetyl. Light and heavy chain cDNA transcription units were linked to DHFR and ADA markers, respectively, and introduced independently into different populations of CHO cells. Pools of transformants expressing either light or heavy chains were independently isolated and selected for resistance to increasing concentrations of MTX or dCF, respectively. Cells expressing both heavy and light chains were obtained by cell hybridization and the resulting hybrids were selected for yet higher levels of resistance to both MTX and dCF. Media from cells expressing heavy or light chain had no 4-hydroxy-3-nitrophenacetyl binding activity, whereas media from hybrid cells contained IgM that exhibited specific hapten binding and complement fixation, and closely resembled the original hybridoma antibody. Cells obtained in this way produced antibody at a rate of approximately 67 I.tg/106 cells/48 hr. According to Wood et al. the ability to select independently for increases in expression of light and heavy chains is advantageous for optimizing the production of functional antibody by the CHO cells.
Genetic modification of the secretory pathway Glucose regulated proteins (GRPs) belong to the class of stress-induced proteins that includes the heat shock proteins. The GRPs are found predominantly in the endoplasmic reticulum as 78 and 94 kD proteins referred to as GRP78 and GRP94. The GRPs have considerable sequence similarity to the heat shock proteins that are found predominantly in the cytoplasm as 70 and 90 kD proteins. The immunoglobulin heavy chain binding protein termed BiP is identical to GRP78; BiP associates with free immunoglobulin heavy chains in the endoplasmic reticulum, and prevents their secretion until they have assembled with light chains. It is believed that GRP78 also associates with improperly folded or underglycosylated proteins and prevents their secretion. Domer et al. [8] have found that secretion of a genetically modified form of tissue-type plasminogen activator (t-PA) that lacks potential glycosylation sites is considerably reduced by its strong association with GRP78. This association is believed to block the transport of the recombinant t-PA from the endoplasmic reticulum to the Golgi apparatus. Domer et al. found that the introduction and amplification of GRP78 antisense constructs resulted in a threefold reduction in GRP78 levels and a corresponding increase in secretion of the recombinant t-PA. Additional studies indicated that in these cells t-PA showed a reduced association with GRP78, and that a greater portion was processed to the mature form and secreted. Two amplifiable markers were used to accomplish the goals of these experiments. DHFR was used as the amplitiable marker to prepare cells producing high levels
Gene amplification in mammalian cells: strategies for protein production Kellems of the recombinant t-PA. Following this, ADA was used as the amplifiable marker to selectively isolate cells with amplified copies of the vector expressing antisense RNA against GRP78 mRNA. In some cases the latter approach resulted in the isolation of cells showing a 10-fold reduction in GRP78 expression. These results illustrate that in cases where an association with GRP78 in the endoplasmic reticulum is inhibitory to secretion, the reduction of GRP78 synthesis by the use of amplified copies of antisense constructs can result in increased secretion of the desired product.
Coexpression of cooperating proteins Factor VIII is an essential participant in the intrinsic pathway of blood coagulation. Hemophilia A is an X-linked bleeding disorder resulting from Factor VIII deficiency. Treatment for this condition has traditionally involved administration of a Factor VIII concentrate derived from human plasma. Although this treatment has merit, serious problems remain, including cost, limited availability, and the significant possibility of life-threatening viral contamination. A major advance occurred with the cloning of the
Factor VIII cDNA. Initial efforts to overproduce Factor VIII cDNA in CHO cells by coamplification with DHFR met with difficulty [9]. The amount of Factor VIII recovered from the media of cells having a 500-fold coamplification of the Factor VIII gene was approximately 2-3 orders of magnitude below that observed when genes encoding other proteins [e.g. erythropoietin, yon Willebrand factor (vWF)] were comparably amplified. One reason fbr the reduced recovery of Factor VIII is the requirement for vWF in the culture medium to promote stable assembly and to provide proteolytic protection of the secreted Factor VIII. Kaufman et al. [10] assessed the requirement for the vWF by deriving cell lines in which Factor VIII and vWF were coamplified. They began with cells in which a Factor VIII expression vector was amplified after linking it with DHFR (Fig. 1). A vWF expression vector linked to ADA was introduced into these cells. Transformants were isolated and coamplification of vWF via linkage to ADA was initiated. At each step in the amplification process the production of vWF and Factor VIII was determined. The results demonstrated that vWF production increases over 200-fold with increasing ADA selection. As
Introduce into DHFR CliO Cells
ilciiV r cDNA
Select for resistance to MTX
DHFR cDNA
@@ MTX resistant cells produce Factor VIII
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...........
cDNA\\\ \~,, ~'~,~r~J~ DHFR ~ - ~ " ' ~
Introduce into Factor \VIII producing I cells
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I resisl F .............
yon Witlebrand factor
Fig. 1. Coexpression of von Willebrand factor (vWF) and Factor Viii eliminates the requirement for exogenously added vWF and increases the yield of Factor VIII. Introduce the vector, pRxPy VIllI and select transfected (MTX-resistant) cells. This produces a low yield of unstable Factor VIII. Cells that co-express both Factor VIII and vWF were obtained by initial transfer and amplification of the Factor VIII expression vector and subsequent transfer and amplification of the vWF expression vector [10]. As the vWF expression vector was amplified over a 200-fold range there was a corresponding increase in the level of Factor VIII activity in serum-free medium.
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Expressionsystems the level of vWF expression increased there was a corresponding increase in the recovery of Factor VIII from serum-free culture medium. The high level of vWF expression achieved through coamplification with ADA replaced the requirement for serum as a source ofvWF and substantially improved the recovery of Factor VIII from genetically engineered mammalian cells.
Highly inducible expression vectors In some cases it will be desirable for a gene of interest to remain inactive during the process of cotransfer and coamplification. This may be necessary because excessive quantities of the gene product may retard cell growth. In other cases the investigator may wish to keep the gene inactive until it is desirable to activate its expression. The latter could be timed to meet specific experimental demands or production requirements. Israel and Kaufman [11] used ADA selection protocols to introduce and amplify a mini-gene encoding the rat glucocorticoid receptor into DHFR-deficient CHO cells. The resulting cells, termed GRA, produce receptor protein at 5-10 times the level found in rat liver or hepatoma cell lines. Because GRA cells are DHFR deficient, they can be used as a host for DHFR based amplification of heterologous genes placed under control of glucocorticoid regulatory elements. In one example, Israel and Kaufman used a bicistronic expression vector containing a glucocorticoid-inducible transcription unit encoding t-PA and a 3' open reading frame encoding DHFR. The vector was introduced into GRA cells and amplified by selection for MTX resistance. At all levels of MTX selection the presence for glucocorticoid resulted in a 5-10 fold increase in t-PA synthesis. Thus, stable inducible expression of amplified genes at relatively high levels can be achieved with this approach.
Double your chances of success The desired gene may not remain faithfully coamplifed with the amplifiable marker as a result of DNA rearrangements and recombination that are known to occur during the amplification process. Because of this it is necessary periodically to isolate and analyze subclones of the selected populations in order to identify those that have faithfully coamplified the nonselectable gene of interest. To reduce the time and effort required for this analysis, Kellems et al. constructed a double selection vector in which a gene of interest, in this case the t-PA gene, is flanked by two amplifiable markers, ADA and DHFR [6]. This plasmid was introduced into DHFR-deficient CHO cells and transformants selected for increased ADA expression. Because ADA is a dominant selectable marker, it is preferable to utilize ADA selection to isolate the initial transformants. These were expanded and altemately selected for increased ADA and DHFR. The level of t-PA expression was monitored as a function of increased selection pressure. The results indicate that a linked gene is more faithfully coamplifed when sandwiched between two amplifiable markers. These findings suggest that the placement of a nonselectable gene between ADA and DHFR may serve as a generally useful strategy for the co-
transfer and coamplification of genes that encode commercially valuable proteins.
Increasing the odds Gene amplification occurs at discrete loci in mammalian cells at frequencies as high as 10-4. The mechanism is not understood but significant efforts are underway to determine the initial events of this process [12,13",14-]. Although the mechanism is not yet understood, genetic and environmental factors have been identified that increase the frequency of gene amplification. Some of these findings present useful techniques for the genetic engineer who is interested in constructing mammalian cells with amplified copies of specific genes.
Genetic elements that increase the frequency of gene amplification Two research groups have identified cis-acting elements that increase the frequency of gene amplification. Stanners and his colleagues [15-.-17"*] have described a middle repetitive element, termed HSAG, that acts in cB to promote amplification of DHFR mini-genes when introduced into a variety of mammalian cell lines. The presence of this element on a DHFR expression plasmid reproducibly increased the production of colonies resistant to 2 btM MTX by 20-200 fold. The DHFR genes in the resulting colonies were amplified 10-20 fold. The dement also enabled the transferred mini-genes to be amplified at a faster ,'ate. The amplification activity was localized to a 1.45 kb fragment that contains Muqike elements, inverted repeats, and A/T-rich regions. Grummt and colleagues [18"] have identified a 370bp element from the non-transcribed spacer region of murine ribosomal RNA genes that increases the copy number of a selectable marker when introduced into recipient cells. The effect on copy number was localized to an A/Trich region at the 5' end of the element and an 11 bp palindrome at its 3' end. Each region interacts with specific cellular proteins. Specific point mutations within the 11 bp palindrome reduced protein/DNA interactions and weakened its ability to stimulate gene amplification. The 11 bp palindrome is conserved in the otherwise strongly divergent, non-transcribed spacer regions of mouse, rat and man, and is also found in the origin/enhancer region of the human papovavirus JC. The mechanisms by which these elements function to promote amplification are not known. McArthur and Stanners [15"] favor a mechanism whereby their element promotes amplification through recombinational mechanisms. Grummt and colleagues [18 "°] favor a replicational mechanism for the element they discovered. Regardless of the mechanism, the existence of cB-regulatory elements that stimulate amplification of linked genes suggests potentially useful applications. Such dements could be included in gene amplification vectors to increase the number of tranformants initially obtained and the rate at which the associated genes are subsequently amplified.
Gene amplification in mammalian cells: strategies for protein production Kellems 727 This could result in saving considerable time and material. Cell lines in which amplification occurs at an unworkably low frequency may become amenable to gene transfer and amplification with the help of these elements.
Cell lines with amplifier phenotypes Gene amplification is a very rare event in normal diploid human and rodent cells [19,20] but it is readily detected in tumors and in permanent cell lines. These findings suggest that genetic changes associated with cellular transformation contribute to the occurrence of gene amplification. Based upon these findings Guilotto, Stark and their colleagues [21] have taken a genetic approach to identify biochemical defects that contribute to the gene amplification process. To do this they selected baby hamster kidney cells with resistance to MTX and N-(phosphonoacetyl)-L-aspartate (PALA) - - an inhibitor of carboamoyl phosphate synthetase, aspartate transcarbamoylase and dihydroorotase (CAD), a multifunctional enzyme catalyzing the first three steps of pyrimidlne metabolism - - and showed that resistance was due to the simultaneous amplification of DHFR and CAD genes. Subsequent analysis showed that the cells that are resistant to MTX and PALA(termed MP cells), showed an increase of as much as 25-fold in the frequency of resistance to coformycin, pyrazofurin, or ouabain (see Table 1 for the enzymes inhibited by these drugs). The pyrozofurin-resistant cells were analyzed and shown to have amplified copies of the UMP synthetase gene. Accordingly, resistance to coformycin and ouabain was presumed to result from amplification of genes encoding AMP deaminase and Na +,K + -ATPase, respectively. The MP cells retained the amplifier phenotype even when resistance to MTX and PALA was lost following extended growth under non-selective conditions. These findings suggested that the MP cells have a heritable change in one or more genes involved in the gene amplification process. Recent phenotypic and genetic analysis of the MP cells revealed that they have a deficiency in the repair of ultraviolet and mitomycin C induced DNA damage [22..]. The amplifier cell yielding the greatest increase in the frequency of gene amplification showed no significant alteration in cell cycle timing. Such cell lines may offer advantages for gene transfer and amplification strategies by reducing the time required to obtain cell lines with amplified copies of desired genes.
Environmental factors affecting gene amplification A number of mutagenic treatments are known to increase the frequency of gene amplification by as much as 10-100 fold [1°°]. In most cases the increase in gene amplification frequency is more than offset by the percentage of cells killed as a result of the mutagenic treatment. Although information regarding the effects of specific mutagenic treatments on gene amplification may prove usefill in understanding the mechanism of this process, such treatments are not routinely used to improve gene transfer and amplification strategies for protein production.
Gene amplification and retrovirus vectors Better titers through amplification Considerable attention is currently devoted to the development of gene therapy protocols for the treatment of certain human genetic disorders. Somatic gene therapy for Factor VIII could entail the introduction of a functional Factor VIII gene into self-renewing hematopoietic stem cells, followed by continual expression of the introduced gene in specific blood cell progeny. Successful accomplishment of this goal would provide a constant and self-renewing source of recombinant Factor VIII and eliminate the need for repeated transfusions of the recombinant protein. Retrovirus vectors are considered to be the most efficient means for introduction of genetic information into recipient cells. The efficiency of gene transfer is often limited by the titer of the replication defective retrovirus vectors utilized in this process. Gene amplification can be very useful to increase viral citer. Following an initial lead provided by Miller et al. [23], who used DHFR amplification to improve viral titers, Israel and Kaufman [24--] have recently reported using coamplification with ADA to improve the citer of replication defective retroviruses capable of transducing Factor VIII. To do this they constructed a retroviral vector encoding Factor VIII and ADA. This construct was introduced into a retroviral packaging cell line and transformants were selected for increased ADA expression. The resulting transformants secreted Factor VIII into the medium and produced replication defective retroviruses capable of transferring Factor VIII and ADA expression to recipient cells. Selection of viral producer cell lines for increased ADA expression yielded cells with a 20-fold increase in both Factor VIII expression and viral citer. These results demonstrate the feasibility of retroviral vector mediated transfer of Factor VIII expression and also the utility of amplifiable markers for increasing viral titer.
Ping-pong amplification without selection Kozak and Kabat [25 ,°] have developed a procedure termed 'ping-pong amplification' by which retrovirus vectors are used to achieve high levels of protein production in mammalian cells. They found that recombinant retroviral vector DNA encoding the human growth hormone (hGH) gene was rapidly amplified when added to cultures containing a mixture of ecotropic (Psi-2) and amphotropic (PAl2 or PA317) packaging cell lines. Under these coculture conditions helper-free virions released from one cell type infect the other, and the result is a theoretically limitless back-and-forth (ping-pong) process of vector transduction between the two cell types. Kozak and Kabat demonstrated that ping-pong vector amplification leads to a high level of hGH production. The amplification process is terminated by isolating individual clones of ecotropic or amphotropic packaging cell lines from the coculture. Stable clones were obtained that produce hGH at levels amounting to 4-6% of the secreted protein. The authors indicate that this method has been used to produce human erythropoietin, the erythropoietin receptor, the erythroblast mitogenic glycoprotein encoded by Friend erythroleukemia virus, the mouse ectopic retro-
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Expressionsystems vires receptor, and a brain protein. The method, therefore, appears to have significant potential for protein production in mammalian cells.
Conclusion T h e ability t o i n t r o d u c e n e w g e n e t i c i n f o r m a t i o n i n t o m a m m a l i a n cells a n d s e l e c t t r a n s f o r m a n t s w i t h desired p r o p e r t i e s offers e n o r m o u s c o m m e r c i a l p o t e n t i a l . T h e ability t o s e l e c t cells t h a t h a v e a m p l i f i e d c o p i e s o f t h e introduced genes adds considerably to the commercial potential o f this p r o c e s s . O n e o f t h e m o s t s u c c e s s f u l strategies f o r o b t a i n i n g a h i g h level o f e x p r e s s i o n o f h e t e r o l o g o u s g e n e s i n m a m m a l i a n cells h a s b e e n b y coamplification of desired genes by genetic linkage with selectable a n d a m p l i f i a b l e m a r k e r s . T h e availability o f a c o l l e c t i o n o f a m p l i f i a b l e g e n e t i c m a r k e r s will p l a y a n i n c r e a s i n g l y i m p o r t a n t r o l e i n a l l o w i n g g e n e t i c e n g i n e e r s to m a n i p u late m a m m a l i a n cells as t h e y w i s h . T h e ability to e n g i n e e r m a m m a l i a n cells p r o d u c i n g l a r g e q u a n t i t i e s o f d e s i r e d RNA a n d p r o t e i n p r o d u c t s h a s o p e n e d u p n e w possibilities o f c o n s i d e r a b l e i m p o r t a n c e t o b i o l o g y a n d m e d i c i n e .
References and recommended reading Papers of special interest, published within the annual period of review, have been highlighted as: • of interest •. of outstanding interest KAUFMANRJ: Selection and Coamplification of Heterologous Genes in Mammalian Cells. Meth Enzymol 1990, 185:537-566. This is one of several very useful reviews dealing with expression of heterologous genes in mammalian cells that appear in this volume of Methods in Enzymology. This article reviews the methods, genetic mark ers, and strategic considerations for using gene amplification to achieve high levels of protein production in mammalian cells. It is a thorough and accurate account of over 10 years of research in this area. 1.
•.
2.
YEUNG C-Y, INGOLtA DE, BOBONIS C, DUNBAR BS, RISER ME, SIC1LIANOMJ, KELLEMSRE: Selective Overproduction of AdenosIne Deaminase in Cultured Mouse Cells. J Biol Chem 1983, 258:8338-8345.
3.
YEUNGC-Y, FRAYNE EG, AL-UBAIDI MR, HOOK AG, INGOLIA DE, BOBONIS C, WRIGHT DR, KELLEMSRE: Amplification and Molecular Cloning of Murine Adenosine Deaminase Gene Sequences. J Biol Chem 1983, 258:15179-15185.
4.
KAUFMANRJ, MURTHAP, INGOIaADE, YEUNG C-Y, KELLEMSRE: Selection and Amplification of Heterologous Genes Encoding Adenosine Deaminase in Mammalian Cells. Proc Natl Acad Sci USA 1986, 83:3136-3140.
5.
INGOLIADE, YEUNG C-Y, ORENGO IF, HARRISON ML, FRAYNE EG, RUDOLPHFB, KELLEMSRE: Purification and Characterization of Adenosine Deaminase from a Genetically Enriched Mouse Cell Line. J Biol Chem 1985, 260:13261-13267.
6.
KELLEMSRE, JOHNSTON EM, WARD KE, LITTLE SP: Adenosine Deaminase: a Dominant Amplifiable Genetic Marker for use in Mamnlalian Cells. In Genetics a n d Molecular Biology of Industrial Microorganisms edited by Hershberger CL, Queener SW, Hegeman G [book]. Washington: American Society for Microbiology 1989, pp 215-225.
7. •.
WOOD CR~ DORNER AJ, MORRIS GE, ALDERMANEM, WILSON D, O'HARA RM, KAUFMANRJ: High Level Synthesis of hnmunoglobulins in Chinese Hamster Ovary Cells. J I m m u n o l 1990, 145:3011-3016. The work presented here shows that a functional antibody can be produced in genetically engineered CHO cells at levels equivalent to or greater than many hybridomas. This was achieved by transfecting different CHO ceils with light and heavy chain expression vectors linked to DHFR and ADA, respectively, for use as an amplifiable marker. Following amplification in independent ceil lines, cell hybrids were obtained that produced functional antibody at a high rate. The expression strategy employed here should prove useful for the production of genetically engineered antibodies and other multi-subunit proteins. 8.
DORNERAJ, KRANE MG, KAUFMANRJ: Reduction of Endogenous GRP78 Levels Improves Secretion of a Heterologous Protein in CHO cells. Mol Cell Biol 1988, 8:4063-4070.
9.
KAUFMANRJ: Genetic Engineering of Factor VIII. Nature 1989, 342:207-208.
10.
KAUFMANRJ, WASLEYLC, DAVIESMV, WISE RJ, ISRAELDI, DORNER AJ: Effect of yon Wlllebrand Factor Coexpression on the Synthesis and Secretion of Factor VIII in Chinese Hamster Ovary Cells. Mol Cell Biol 1989, 9:1233-1242.
11.
ISRAELDI, KAUFMANNRJ: Highly Inducible Expression from Vectors Containing Multiple GREs in CHO Cells Overexpressing the Glucocorticoid Receptor. Nucleic Acids Res 1989, 17:4589-4604.
12.
TRASKBJ, HAMIaNJL: Early Dihydrofolate Reductase Gene Amplification Events in CHO Cells Usually Occur on the Same Chromosome Arm as the Original Locus. Genes Dev 1989, 3:1913-1925.
13. .
SMITHKA, GORMANPA, STARKMB, GROVESRP, STARKGI~ Distinctive Chromosomal Structures are Formed Very Early in the Amplification of CAD Genes in Syrian Hamster Cells. Cell 1990, 63:1219-1227. Fluorescence in situ hybridization to metaphase chromosomes was used to visualize recently amplified arrays of CAD genes. The results indicate that very large regions of DNA, each as long as 10kb, are tandemly arrayed near one of the original CAD genes that remained at its original location. The tandem arrays of amplified DNA are believed to arise via sister chromatid exchange. 14. •
W1NDLEB, DRAPER BW, YIN Y, O'GORMAN S, WAHL GM: A Central Role for Chromosome Breakage in Gene Amplification, Deletion Formation, and Amplification Integration. Genes Dev 1991, 5:160-174. Like [13"], this paper also reports fluorescence in situ hybridization to examine the early products generated during amplification of the single DHFR locus present in a line of CHO ceils. Windle et al. show that cells having undergone DHFR gene amplification are prone to deletion of the DHFR gene and flanking sequences at the original locus. These findings, in contrast to those presented in [12] and [13"], implicate chromosome breakage as an early or initial event in the amplification process. 15. .,
MCARTHURJG, STANNERS CP: A Genetic Element that Increases the Frequency of Gene Amplification. J Biol Chem 1991, 266:6000~005. In this manuscript Mcarthur and Stanners show that a mammalian repetitive sequence element, HSAG-1, can act in cis, to increase the frequency of amplification of a vector following gene transfer into mammalian cells, the s~nulation of amplification was as much as 200 fold and the effect was observed in a variety of mammalian ceils lines. The genetic element thus identified could be a useful addition to mammalian gene amplification vectors. 16. ,.
BRITEL LK, MCARTHUR JG, STANNERS CP: Sequence Requirments for the Stimulation of Gene Amplification bya Mammalian Genomic Eloment. Gene 1991, 102:149-156. Deletion analysis of the HSAG-1 element referred to in [ 15"'] revealed the presence of multiple positive acting elements that are required for the amplification effect, a highly active 1,45 kb fragment was identified that contains the necessary sequence elements to promote gene am-
Gene amplification in mammalian cells: strategies for protein production Kellems 729 plitication following transfer into mammalian cells. Additional HSAG-1 like elements were tested and found to stimulate vector amplification following introduction into mammalian cells.
lected loci. Here they associate the amplifier phenotype with an altered DNA repair capability. These, or related cell lines, may prove useful as recipients in gene transfer and amplification strategies.
17. °°
23.
MCARTHURJG, BE1TEL LK, CHAMBERLAIN J, STANNERS CP: Elements w h i c h Stimulate G e n e Amplification in Mammalian Cells: Role of Recombinogenic Sequences/Structures and Transcriptional Activities. Nucleic Acids Res 1991, 19:2477-2484. Structural and Functional analysis of the HSAG-1 regulating elements revealed that the ability to stimulate amplification is orientation dependent and that the element must be transcribed in order to exert its effect. This study provides important information that is critical for proper use of these elements as components of mammalian gene amplification vectors. 18. oo
WEGNERM, SCHWENDER S, DINKL E, GRUMMT F: Interaction of a Protein with a Palindromic Sequence from Murine rDNA Increases t h e O c c u r r e n c e of Amplification-dependent Transformation in Mouse Cells. J Biol Cbem 1990, 265:13925-13932. An l l b p palindrome is described that stimulates by more than 10-fold the rate of transformation dependent amplification of a selectable marker gene. The palindromic sequence interacts specifically with proteins from mouse extracts. Mutations that reduce protein binding also reduce the stimulation of gene amplification. The sequence elements identified by these investigators may play a useful role as a component of gene amplification vectors. 19.
WRIGHTJA, SMITH HS, WATT FM, HANCOCK MC, HUDSON DL, STARK GR: DNA Amplification is Rare in Normal H u m a n Cells. Proc Natl Acad Sci USA 1990, 87:1791-1795.
20.
TLSTYTD: Normal Diploid H u m a n and Rodent Cells Lack a Detectable Frequency of Gene Amplification. Proc Natl Acad Sci USA 1990, 87:3132-3136.
21.
22. °°
GIULOTTOE, KNIGhtS C, STARK GR: Hamster Cells with Increased Rates of DNA Amplification, a N e w Phenotype. Cell 1987, 48:837-845.
GIULOTTOE, BERTONI L, ATTOLINI C, RANIALD1G, ANGLANAM: BHK Cell Lines with Increased Rates of Gene Amplification are Hypersensitive to Ultraviolet Light. Proc Natl Acad Sci USA 1991, 88:3484-3488. These authors had previously isolated a cell line [21] that is character ized by a 25-fold increase in the frequency of gene amplification at se-
MILLERAD, LAW M-F, VERMA IM: Generation of Helper-free Amphotropic Retroviruses that T r a n s d u c e a Dominant-acting, Methotrexate-resistant Dihydrofolate Reductase Gene. Mol Cell Biol 1985, 5:431-437.
24. ..
ISRAELDI, KAUFMANFJ: Retroviral-mediated Transfer and Amplification of a Functional H u m a n Factor VIII Gene. B/ood 1990, 75:1074-1080. Findings reported here demonstrate the feasibility of retroviral mediated transfer of human Factor VIII and the utility of gene amplification for increasing the viral titer. 25. •.
KOZAKSL, KABAT D: Ping-pong Amplification of a Retroviral Vector Achieves a High-level G e n e Expression: H u m a n Growth H o r m o n e Production. J Virol 1990, 64:3500-3508. These authors show that when retroviral vector DNA is added to cocultures that contain packaging cell lines with different envelope specificities, each cell type produces helper-free virions that infect the other. This ping-pong cycle of vector transduction leads to amplification of proviral sequences and a high level production of encoded proteins. 26.
LEHRMAN MA, ZHU X, KHOUNLO S: Amplification and Molecular Cloning of t h e H a m s t e r Tunicamycin-sensitive N-Acetylglucosamine-l-Phosphate Transferase Gene. T h e Hamster and Yeast Enzymes Share a C o m m o n Peptide Sequence. J Biol Cbem 1988, 263:19796-19803.
27.
KONTISKJ, ARFIN SM: Isolation of a cDNA Clone for H u m a n Threonyl-tRNA Synthetase: Amplification of the Structural G e n e in Borrelidin-resistant Cell Lines. Mol Cell Biol 1989, 9:1832-1838.
28.
TsuI FWL, ADRULISIA, MURIALDOH, SIMINOVITCHL: Amplitication of the Gene for Histidyl-tRNA Synthetase in Histidinol Resistant Chinese Hamster Ovary Cells. Mol Cell Biol 1985, 5:2381-2388.
29.
PAUWPG, JOHNSON MD, MOORE P, MORGAN M, F1NEMANRM, KALKAT, ASH JF: Stable Gene Amplification and Overexpression of Sodium- and Potassium-activated ATPase in Hela Cells. Mol Cell Biol 1986, 6:1164-1171.
RE Kellems, Department of Biochemistry and Institute for Molecular Genetics, Baylor College of Medicine, Houston, Texas 77030, USA.
