Use of mammalian cell expression cloning systems to identify genes for cytokines, receptors, and regulatory proteins Naoki Nakayama, Takashi Yokota and Ken-ichi Arai Institute of M e d i c a l Science, The University of Tokyo, Japan Mammalian cell expression cloning has become a standard technique for the isolation of mammalian genes or cDNAs. Its advantage is that the biological functions of the gene of interest are used for cloning. Therefore, the identified cDNAs or genes should be functional in vivo, and there is no need for physical or chemical information about the gene products, so that protein purification in sufficient quantity to raise antibodies or to obtain amino acid sequences is not necessary. Here, we summarize recent progress in mammalian cell cloning systems, and discuss the possible directions in which this technique will lead. Current Opinion in Biotechnology 1992, 3:497-505
Introduction A gene of interest can be identified based on the structure or function of its protein product. The cloning procedure that involves transfer of a particular phenotype of one cell to another by transfection of a chromosomal gene or cDNA is termed expression cloning. There are several considerations in developing an expression cloning protocol. A choice has to be made between the use of genomic DNA or a cDNA, between transient transfection or stable transfection protocols, and the type of recipient cell, such as fibroblasts, hematopoietic cells, lymphocytes, and embryonic stem cells must be selected. The growth characteristics of recipient cells (growth facto>dependent, or transformed), the choice of vectors-(episomal or integrative) and expression devices are also important factors. Expression cloning of a mammalian gene using mammalian cells as recipients was employed initially to isolate cellular oncogenes. The necessity for a mammalian cloning system was straightforward in this case, as the assay to clone the genes depended on their in vivo function, namely the ability of the e n c o d e d proteins to transform the phenotype of a recipient mammalian cell line. The ras oncogene is a successful example. It was cloned by direct transfection of the NIH3T3 cell line with chromosomal DNA derived from cancerous ceils. The advantage of this m e t h o d is that because recipient ceils take up large pieces of chromosomal DNA, the number of resultant stable transfectants is within a reasonable range for screening purposes. Limitations to this m e t h o d lie in the later steps of the procedure.
To enrich a particular gene of interest from the transfected DNA integrated into the chromosome of recipient ceils, transfection of the recipient cells with the chromosomal DNA of transformants is required. In order to narrow d o w n the protein-coding region within the transfected DNA, this process must be repeated. Direct transfection of 10T1/2 fibroblasts with a de-methylated, whole cosmid genomic library, and visual inspection for myoblast-like colonies has identified a myogenic gene, m y d [1]. Cosmid vectors can reduce the size of integrated DNA segments to 40-50 kb. A problem still remains, however, in that the proteincoding region of a gene is generally spliced into small pieces and scattered around the genome, so that determining the primary structure of the coded product may take a considerable length of time. An alternative method that allows protein-coding sequences to be cloned directly is the expression cloning of cDNAs. Development of a protocol to ensure that the entire coding regions of mRNAs are converted into cDNA was obligatory for cDNA expression cloning. As cDNAs do not contain transcriptional signals themselves, addition of a correct promoter/enhancer, a splicing site, and a poly(A) signal is necessary for cDNAs to be expressed [2]. The frequency of cloning a particular cDNA depends on the mRNA levels in the cells from which the cDNA library was constructed. The transfection efficiency of recipient cells is crucial to ensure statistically that a rare cDNA of interest has been transfected successfully. When the assay involves a mixture of transfectants, detecting a signal of one cDNA over the background also depends on the ex-
Abbreviations ARS--autonomously replicating sequences; BPV--bovine papilloma virus; CEN--centromere DNA; CMV---cytomegalovirus; DEAE--diethylaminoethyl; EBV--Epstein-Barr virus; FACS~fluorescence-activated cell sorting; G-CSF--granulocyte colony stimulating factor; GM-CSF--granulocyte-macrophage colony stimulating factor; IL--interleukin; LIF--leukemia inhibitory factor; orb--origin of replication; PMS--plasmid maintenance sequence; RSV--Rous sarcoma virus; SV--simian virus. © Current Biology Ltd ISSN 0958-1669
497
498 Expressionsystems pression level of each cDNA in the recipient cells. Therefore, over the last decade, maximizing both expression levels of each cDNA in the recipient cells and the transfection efficiency of these cells has b e e n the main consideration. Stable transfection is usually more efficient with the electroporation method than the calcium phosphate precipitation method. The diethylaminoethyl (DEAE) dextran method is highly efficient, but often inhibits long term growth. Therefore, it is used most successfully for transient transfection. Michael Kriegler [3] c o m p a r e d the strengths of different types of promoters in a transient transfection assay of several different cell lines, and concluded that SR(x is the strongest. Other popular promoters for cDNA cloning vectors are the simian virus (SV) 40 early/late p r o m o t e r [2], Rous sarcoma virus (RSV) long terminal repeat (LTR), cytomegalovirus (CMV) promoter [4], and adenovirus late promoter [5]. SRcz is basically a modified SV40 early p r o m o t e r with the R/U5 segment of h u m a n T-cell leukemia virus LTR inserted immediately d o w n s t r e a m of it [6].
Expression cloning of cytokines and their receptors Cytokine cDNA cloning Expression cloning of cDNAs was first employed to isolate cytokine cDNAs b y Arai and colleagues [7,8]. Cytokines are generally c o m p o s e d of a single p o l y p e p tide, and secreted from T lymphocytes. If transfection efficiency is high enough to accommodate part of a cDNA library, and expression levels of individual cDNAs are also high, the activity of a particular cytokine cDNA product is detectable in a s u p e m a t a n t of total transfected cells. By subdividing the cDNA library repeatedly and transfecting recipient ceils over several cycles (sibling) , the single cDNA responsible for a particular cytokine activity can be identified. Stable transfection is not necessary for this screening method, which saves a substantial length of time. The principles of this m e t h o d are illustrated in Fig. 1. For this method, COS7 ceils are often used as recipient cells in combination with a vector containing the SV40 replication origin. COS7 is a m o n k e y kidney cell line expressing the SV40 large T antigen that induces rapid, origin-dependent replication of the transfected plasmids. This amplifies expression levels of individual cDNAs through the gene dosage effect and improves the promoters that drive expression of cDNAs. These ensure that cytokines expressed in the COS7 cell supernatant will be detected even if the cytokine cDNA is relative rare in the population. Analogous cell systems in mice are COP and WOP. These cells express the p o l y o m a large T antigen so that a vector containing the p o l y o m a replication origin can be used in the same way. This is particularly useful w h e n COS7 ceils are sensitive to the cytokine of interest, or express an activity that cross-reacts with indicator ceils. The classical SV40 early promoter was used to clone interleukin (IL)-2, IL-3, granulocyte-macrophage colony stimulating factor (GM-CSF), and IL-4 [7-9]. The improved
SV40 early promoter, SR0t, was used successfully to clone IL-5, IL-10, and h u m a n IL-3 from an activated T-cell cDNA library [7,9,10]. The adenovirus major late promoter with a tripartite leader sequence c o m b i n e d with the VA gene allowed GM-CSF, h u m a n IL-3, and IL-11 to be cloned [5,9,11].
Cloning of cytokine receptor cDNAs Recent progress in expression cloning of m a m m a l i a n cDNAs has p e a k e d with the invention of a n u m b e r of sophisticated methods for the detection of cDNAs of interest. This is especially evident in cytokine receptor cDNA cloning. Receptor cDNAs have b e e n cloned b y essentially the same m e t h o d as cytokine cDNAs. Here, cDNA cloning of so-called type-I cytokine receptors is used as an example [12"]. Receptor mRNAs are generally larger than cytokine mRNAs, and expression levels of receptor genes are m u c h lower c o m p a r e d to most of the cytokine genes. Therefore, the receptor mRNAs have often b e e n enriched. Cells expressing a particular receptor at high levels are enriched b y fluorescence-activated cell sorting (FACS) [13], and cDNAs of a certain length (size-selected cDNAs) are purified before library construction. The most straightforward m e t h o d for receptor cloning is the sibling m e t h o d as described above. The cDNA library is subdivided, COS7 ceils are transfected with each pool, and ligands labeled with 125I bind to individual transfected cell mixtures (Fig. 2). If the receptor cDNA is c o m m o n in a subgroup, binding of the ligand can be detected a b o v e the background. Repetition of subgrouping and transfection would reveal a single cDNA responsible for ligand binding. Mouse erythropoietin receptor [14], granulocyte colony stimulating factor (G-CSF) receptor [15] and IL-3 receptor (x-chain [16"] were successfully isolated b y this method. A convenient screening method for receptor cloning was invented originally for isolating T-cell surface marker cDNAs [4]. This method, called panning, needs receptor ligands or specific antibodies to be immobilized on tissue culture plates (Fig. 2). COS7 ceils are transiently transfected with a size-selected and often subgrouped cDNA library, and then each mixture of the transfected cells is placed on a plate that has b e e n precoated ~with ligands or antibodies. Unattached ceils, representing those that do not express receptors, are removed, and plasmid DNAs retained in the attached ceils are recovered into Escherichia coil Transient transfection and panning are repeated to enrich a set of specific cDNA clones. The individual cDNA is finally transferred to COS7 cells and binding of the ligand and/or antibody to the ceils is confirmed. This m e t h o d has b e e n used successfully to clone h u m a n IL-2 receptor B-chain [17], m o u s e IL-3 receptor [3-chain [18], m o u s e IL-3/GM-CSF/IL-5 receptor ~-chain [19], m o u s e IL-5 receptor (z-chain [20], mouse IL-4 receptor [21], and rat ciliary neurotrophic factor receptor [22"']. Of these, cloning for the IL-4 and ciliary neurotrophic factor receptor cDNAs e m p l o y e d the ligand-panning method. Expression of a single chain of a multi-chain receptor often gives low affinity binding with the correspond-
Use of mammalian cell expression cloning systems Nakayama, Yokota and Arai
mRNA from cytokine producing cells
SV40 ori/early promoter (SRo0
16S/19S intron
>
#
cDNA library
~
H
~
poly A SV40
Subdividing
Transfection
~
Amplification Transient expression
Z
Collect supernatant
Bioassay
Positives
DNA recovery
J Fig. 1. Principles of cytokine cDNA expression cloning. A cDNA library is constructed in a vector that allows each clone to express a cDNA and to be amplified in COS7 cells. The library is subdivided and each cDNA is introduced into COS7 cells. Cytokine activity is assayed in the supernatant. Positive cDNA pools are further subdivided and the process is repeated until an individual cDNA responsible for cytokine activity can be detected and isolated. ing ligand. The ligand-panning method is likely to be ineffective in this case. As the low affinity is usually a result of the fast dissociation rate of the ligand, attachment of the receptor-expressing cells to the ligand-immobilized tissue culture plates will be unstable. An alternative m e t h o d involves FACS of the COS7 ceils transiently transfected with a cDNA library, which is also illustrated in Fig. 2. Ligands labeled with biotin, or specific antibodies for the receptor of interest are used for sorting. A fraction of cells that express the receptor is collected and the retained plasmids are recovered. Repeated transfection and FACS will enrich the population of receptor cDNAs. The IL-6 receptor cDNA clone was identified by this m e t h o d [23].
The fourth method for cloning rare receptor cDNAs is the emulsion method (Fig. 2). This involves transfection of COS7 cells with subgrouped cDNA libraries, culturing ceils on chamber slides, binding of labeled ligands (usually with 125I) to the cells, and autoradiography with a photographic emulsion. Direct inspection under a microscope allows even a single positive cell to be detected. Repetition of subgrouping and transfection of positive cDNA pools leads to a single cDNA clone. Mouse and h u m a n GM-CSF receptor et-chains [24,25"], h u m a n IL-3 receptor a-chains [16"',26"], hum a n IL-7 receptor [27], h u m a n G-CSF receptor [28] and h u m a n leukemia inhibitory factor (LIF) receptor [29"] have b e e n cloned using this method.
499
500
Expression systems
~
Receptor-bearing cells
mRNA V40ori
rlo0sm/%eJnt, Vn U 5* cDNA library
SV40 polyA
D'cosz ce,s
Introduction A gene of interest can be identified based on the structure or function of its protein product. The cloning procedure that involves transfer of a particular phenotype of one cell to another by transfection of a chromosomal gene or cDNA is termed expression cloning. There are several considerations in developing an expression cloning protocol. A choice has to be made between the use of genomic DNA or a cDNA, between transient transfection or stable transfection protocols, and the type of recipient cell, such as fibroblasts, hematopoietic cells, lymphocytes, and embryonic stem cells must be selected. The growth characteristics of recipient cells (growth facto>dependent, or transformed), the choice of vectors-(episomal or integrative) and expression devices are also important factors. Expression cloning of a mammalian gene using mammalian cells as recipients was employed initially to isolate cellular oncogenes. The necessity for a mammalian cloning system was straightforward in this case, as the assay to clone the genes depended on their in vivo function, namely the ability of the e n c o d e d proteins to transform the phenotype of a recipient mammalian cell line. The ras oncogene is a successful example. It was cloned by direct transfection of the NIH3T3 cell line with chromosomal DNA derived from cancerous ceils. The advantage of this m e t h o d is that because recipient ceils take up large pieces of chromosomal DNA, the number of resultant stable transfectants is within a reasonable range for screening purposes. Limitations to this m e t h o d lie in the later steps of the procedure.
To enrich a particular gene of interest from the transfected DNA integrated into the chromosome of recipient ceils, transfection of the recipient cells with the chromosomal DNA of transformants is required. In order to narrow d o w n the protein-coding region within the transfected DNA, this process must be repeated. Direct transfection of 10T1/2 fibroblasts with a de-methylated, whole cosmid genomic library, and visual inspection for myoblast-like colonies has identified a myogenic gene, m y d [1]. Cosmid vectors can reduce the size of integrated DNA segments to 40-50 kb. A problem still remains, however, in that the proteincoding region of a gene is generally spliced into small pieces and scattered around the genome, so that determining the primary structure of the coded product may take a considerable length of time. An alternative method that allows protein-coding sequences to be cloned directly is the expression cloning of cDNAs. Development of a protocol to ensure that the entire coding regions of mRNAs are converted into cDNA was obligatory for cDNA expression cloning. As cDNAs do not contain transcriptional signals themselves, addition of a correct promoter/enhancer, a splicing site, and a poly(A) signal is necessary for cDNAs to be expressed [2]. The frequency of cloning a particular cDNA depends on the mRNA levels in the cells from which the cDNA library was constructed. The transfection efficiency of recipient cells is crucial to ensure statistically that a rare cDNA of interest has been transfected successfully. When the assay involves a mixture of transfectants, detecting a signal of one cDNA over the background also depends on the ex-
Abbreviations ARS--autonomously replicating sequences; BPV--bovine papilloma virus; CEN--centromere DNA; CMV---cytomegalovirus; DEAE--diethylaminoethyl; EBV--Epstein-Barr virus; FACS~fluorescence-activated cell sorting; G-CSF--granulocyte colony stimulating factor; GM-CSF--granulocyte-macrophage colony stimulating factor; IL--interleukin; LIF--leukemia inhibitory factor; orb--origin of replication; PMS--plasmid maintenance sequence; RSV--Rous sarcoma virus; SV--simian virus. © Current Biology Ltd ISSN 0958-1669
497
498 Expressionsystems pression level of each cDNA in the recipient cells. Therefore, over the last decade, maximizing both expression levels of each cDNA in the recipient cells and the transfection efficiency of these cells has b e e n the main consideration. Stable transfection is usually more efficient with the electroporation method than the calcium phosphate precipitation method. The diethylaminoethyl (DEAE) dextran method is highly efficient, but often inhibits long term growth. Therefore, it is used most successfully for transient transfection. Michael Kriegler [3] c o m p a r e d the strengths of different types of promoters in a transient transfection assay of several different cell lines, and concluded that SR(x is the strongest. Other popular promoters for cDNA cloning vectors are the simian virus (SV) 40 early/late p r o m o t e r [2], Rous sarcoma virus (RSV) long terminal repeat (LTR), cytomegalovirus (CMV) promoter [4], and adenovirus late promoter [5]. SRcz is basically a modified SV40 early p r o m o t e r with the R/U5 segment of h u m a n T-cell leukemia virus LTR inserted immediately d o w n s t r e a m of it [6].
Expression cloning of cytokines and their receptors Cytokine cDNA cloning Expression cloning of cDNAs was first employed to isolate cytokine cDNAs b y Arai and colleagues [7,8]. Cytokines are generally c o m p o s e d of a single p o l y p e p tide, and secreted from T lymphocytes. If transfection efficiency is high enough to accommodate part of a cDNA library, and expression levels of individual cDNAs are also high, the activity of a particular cytokine cDNA product is detectable in a s u p e m a t a n t of total transfected cells. By subdividing the cDNA library repeatedly and transfecting recipient ceils over several cycles (sibling) , the single cDNA responsible for a particular cytokine activity can be identified. Stable transfection is not necessary for this screening method, which saves a substantial length of time. The principles of this m e t h o d are illustrated in Fig. 1. For this method, COS7 ceils are often used as recipient cells in combination with a vector containing the SV40 replication origin. COS7 is a m o n k e y kidney cell line expressing the SV40 large T antigen that induces rapid, origin-dependent replication of the transfected plasmids. This amplifies expression levels of individual cDNAs through the gene dosage effect and improves the promoters that drive expression of cDNAs. These ensure that cytokines expressed in the COS7 cell supernatant will be detected even if the cytokine cDNA is relative rare in the population. Analogous cell systems in mice are COP and WOP. These cells express the p o l y o m a large T antigen so that a vector containing the p o l y o m a replication origin can be used in the same way. This is particularly useful w h e n COS7 ceils are sensitive to the cytokine of interest, or express an activity that cross-reacts with indicator ceils. The classical SV40 early promoter was used to clone interleukin (IL)-2, IL-3, granulocyte-macrophage colony stimulating factor (GM-CSF), and IL-4 [7-9]. The improved
SV40 early promoter, SR0t, was used successfully to clone IL-5, IL-10, and h u m a n IL-3 from an activated T-cell cDNA library [7,9,10]. The adenovirus major late promoter with a tripartite leader sequence c o m b i n e d with the VA gene allowed GM-CSF, h u m a n IL-3, and IL-11 to be cloned [5,9,11].
Cloning of cytokine receptor cDNAs Recent progress in expression cloning of m a m m a l i a n cDNAs has p e a k e d with the invention of a n u m b e r of sophisticated methods for the detection of cDNAs of interest. This is especially evident in cytokine receptor cDNA cloning. Receptor cDNAs have b e e n cloned b y essentially the same m e t h o d as cytokine cDNAs. Here, cDNA cloning of so-called type-I cytokine receptors is used as an example [12"]. Receptor mRNAs are generally larger than cytokine mRNAs, and expression levels of receptor genes are m u c h lower c o m p a r e d to most of the cytokine genes. Therefore, the receptor mRNAs have often b e e n enriched. Cells expressing a particular receptor at high levels are enriched b y fluorescence-activated cell sorting (FACS) [13], and cDNAs of a certain length (size-selected cDNAs) are purified before library construction. The most straightforward m e t h o d for receptor cloning is the sibling m e t h o d as described above. The cDNA library is subdivided, COS7 ceils are transfected with each pool, and ligands labeled with 125I bind to individual transfected cell mixtures (Fig. 2). If the receptor cDNA is c o m m o n in a subgroup, binding of the ligand can be detected a b o v e the background. Repetition of subgrouping and transfection would reveal a single cDNA responsible for ligand binding. Mouse erythropoietin receptor [14], granulocyte colony stimulating factor (G-CSF) receptor [15] and IL-3 receptor (x-chain [16"] were successfully isolated b y this method. A convenient screening method for receptor cloning was invented originally for isolating T-cell surface marker cDNAs [4]. This method, called panning, needs receptor ligands or specific antibodies to be immobilized on tissue culture plates (Fig. 2). COS7 ceils are transiently transfected with a size-selected and often subgrouped cDNA library, and then each mixture of the transfected cells is placed on a plate that has b e e n precoated ~with ligands or antibodies. Unattached ceils, representing those that do not express receptors, are removed, and plasmid DNAs retained in the attached ceils are recovered into Escherichia coil Transient transfection and panning are repeated to enrich a set of specific cDNA clones. The individual cDNA is finally transferred to COS7 cells and binding of the ligand and/or antibody to the ceils is confirmed. This m e t h o d has b e e n used successfully to clone h u m a n IL-2 receptor B-chain [17], m o u s e IL-3 receptor [3-chain [18], m o u s e IL-3/GM-CSF/IL-5 receptor ~-chain [19], m o u s e IL-5 receptor (z-chain [20], mouse IL-4 receptor [21], and rat ciliary neurotrophic factor receptor [22"']. Of these, cloning for the IL-4 and ciliary neurotrophic factor receptor cDNAs e m p l o y e d the ligand-panning method. Expression of a single chain of a multi-chain receptor often gives low affinity binding with the correspond-
Use of mammalian cell expression cloning systems Nakayama, Yokota and Arai
mRNA from cytokine producing cells
SV40 ori/early promoter (SRo0
16S/19S intron
>
#
cDNA library
~
H
~
poly A SV40
Subdividing
Transfection
~
Amplification Transient expression
Z
Collect supernatant
Bioassay
Positives
DNA recovery
J Fig. 1. Principles of cytokine cDNA expression cloning. A cDNA library is constructed in a vector that allows each clone to express a cDNA and to be amplified in COS7 cells. The library is subdivided and each cDNA is introduced into COS7 cells. Cytokine activity is assayed in the supernatant. Positive cDNA pools are further subdivided and the process is repeated until an individual cDNA responsible for cytokine activity can be detected and isolated. ing ligand. The ligand-panning method is likely to be ineffective in this case. As the low affinity is usually a result of the fast dissociation rate of the ligand, attachment of the receptor-expressing cells to the ligand-immobilized tissue culture plates will be unstable. An alternative m e t h o d involves FACS of the COS7 ceils transiently transfected with a cDNA library, which is also illustrated in Fig. 2. Ligands labeled with biotin, or specific antibodies for the receptor of interest are used for sorting. A fraction of cells that express the receptor is collected and the retained plasmids are recovered. Repeated transfection and FACS will enrich the population of receptor cDNAs. The IL-6 receptor cDNA clone was identified by this m e t h o d [23].
The fourth method for cloning rare receptor cDNAs is the emulsion method (Fig. 2). This involves transfection of COS7 cells with subgrouped cDNA libraries, culturing ceils on chamber slides, binding of labeled ligands (usually with 125I) to the cells, and autoradiography with a photographic emulsion. Direct inspection under a microscope allows even a single positive cell to be detected. Repetition of subgrouping and transfection of positive cDNA pools leads to a single cDNA clone. Mouse and h u m a n GM-CSF receptor et-chains [24,25"], h u m a n IL-3 receptor a-chains [16"',26"], hum a n IL-7 receptor [27], h u m a n G-CSF receptor [28] and h u m a n leukemia inhibitory factor (LIF) receptor [29"] have b e e n cloned using this method.
499
500
Expression systems
~
Receptor-bearing cells
mRNA V40ori
rlo0sm/%eJnt, Vn U 5* cDNA library
SV40 polyA
D'cosz ce,s
