METHOD FOR ELECTRICALLY IMMORTALIZING LYMPHOID CELLS

Abstract: A method for electrically immortalizing lymphoid cell lines through the introduction of cloned cellular oncogenes by high voltage electric pulses. A human B-lymphocyte suspension is placed between two electrodes. Naked DNA containing a cellular oncogene, preferably human c-myc, is added to the cell suspension. An electric potential is applied to the electrodes in a pulse of from about 0.1 nanosecond to 10 milliseconds to create an electric field of about 1 to about 40 kV/cm, thereby introducing the oncogene into at least some of the cells to immortalize those cells. Also disclosed are immortalized B-lymphoid cells containing an exogenous, immortalizing cellular oncogene.

Description: METHOD FOR ELECTRICALLY IMMORTALIZING LYMPHOID CELLS Background of the Invention This invention relates to a method for preparing immortalized lymphoid cell lines through the introduction of cloned cellular oncogenes by high voltage electric fields, and to immortalized lymphoid cells prepared by that method. Most animal cells, when cultured in vitro, grow poorly or not at all. Such cell cultures usually exhibit density-dependent growth, a phenomenon that limits cellular reproduction as cell density increases. In addition, most human cells lose the ability to grow after approximately 50 generations of in vitro culture. Moreover, cells such as antibody-producing human B- lymphocytes, ordinarily produce large quantities of antibody only transiently and in response to certain external stimuli. Such cells tend to lose the ability to produce the protein desired when cultured in vitro. Immortalized cells, on the other hand, have the ability to grow indefinitely in vitro. These immortalized cells often retain the ability to produce specialized proteins, such as im unoglobulins. A culture grown from a single immortalized antibody-producing cell produces a monoclonal antibody. Human lymphoid cells have proven to be remarkably recalcitrant to immortalization by many of the techniques that have been successful in rodent and other models. Several techniques are currently in use for immortalizing lymphoid cells to produce monoclonal antibodies in culture. Perhaps the most ' widely used technique is hybridization of the lymphocyte with a transformed cell line, as described generally in G. Kohler & C. Milstein, Nature 526: 495-497 (1975) and European Journal of Pharmacology 6: 511-519 (1976). These references describe a technique whereby a myeloma or virus-transformed cell line is fused with a B-lymphocyte population. The transformed line contributes immortality (i.e., the ability to grow indefinitely in culture) to the hybrid. The B-lymphocyte contributes the ability to make the light and heavy chains of the desired antibody to the hybrid. Such fusions have generally been performed using polyethylene glycol or dextran sulfate as mediators. The resulting hybrids (hybridomas) are separated and grown in individual cultures which individually produce monoclonal antibody. Another fusion technique that has recently been employed is electrofusion. In electrofusion the two types of parental cells are placed in a non-conducting isoosmotic environment. An alternating current is used to electrically align the cells to be fused. The membranes are compressed, and fusion occurs when the cells are subjected to brief pulses of high voltage direct current pulses. See, e.g., U. Zimmerman & J. Greyson, "Electric Field-Induced Cell Fusion", Biotechniques, September/October 1983: 118-122. These fusion techniques have significant limitations. First, immortality of the antibody-producing cells is the result of the introduction of a vast amount of genetic information, only a minute amount of which is required for growth and immortality. Thus, although only one, or at most, a few genes are responsible for the immortality of the resulting hybridoma, virtually all the genetic material of the immortal parent is transferred to the hybrid. Another disadvantage of the fusion technique is that fusion is not selective, and unproductive cell fusion occurs between either of the two parents and between more than two cells. Another method for immortalizing human lymphocytes is Epstein Barr Virus (EBV) transformation and RNA tumor virus transformation. See, e.g., N. Brown & G. Miller, "Immunoglobulin Expression by Human B-Lymphocytes Clonally Transformed by Epstein Barr Virus", J. Immunology 128: 24- 29 (1982). In this method, the human lymphocyte is infected with a B-cell-specific virus. The viral transformation leads to immortalization and growth of the ■ cells. A disadvantage of this method is the possible synthesis of contaminating viral products as well as the virus itself and, as yet, little antibody synthesis. Also, current data demonstrate that long-term growth resulting from EBV infection is the result of only a small amount of the viral genome. Recently the viral DNA markers have been eliminated from antibody-producing hybrids by EBV infection of the antibody-producing cells followed by a subsequent fusion of this cell with a hybrid line created from a previous human B-cell-mouse myeloma fusion. The resulting hybrid cells do not contain EBV DNA and contain little of the mouse genome, but do make specific amounts of human antibody. While it is clear that the foregoing methods can produce a long-term hybrid cell line that produces antibody, these techniques all use a tremendous overload of genetic information to achieve the desired immortalized cell line. This excess genetic baggage can lead to instability of the lines. There are other techniques currently in use for the introduction of genes into cells. The most common method is CaPO^ transfection, wherein the desired gene is precipitated onto the cell surface and then phagocytized or otherwise incorporated into the cell. The human B- lymphocyte and ly phoblasts have proven to be recalcitrant to CaPO^ transfection procedures. To this end other methods have been employed; for example, enclosing the genes of interest into artificial lipid bags (liposomes) followed by fusion with polyethyleneglycol or by electrofusion. Another technique in use is direct injection of the genes into the cell with the aid of microscopes and micromanipulators. These methods are laborious and success has been relatively limited. Finally, an improvement relating to transfection or injection techniques has been the construction of retrovirus vectors such as RNA tumor viruses which have been genetically manipulated so that they carry the gene of interest but are unable to reproduce themselves. The viruses used can infect and stably integrate with the human B-lymphoblast genome. The use of this method, while a great improvement over those described above, has received criticism for several reasons. First, the viruses are produced in the presence of a helper virus which could conceivably lead to continued propagation and reinfection of cells. In addition, cells may contain latent, quiescent, nonreplicative virus of unknown origin and virulence. The presence of RNA virus genome from the infecting virus could conceivably cause these latent or nonreplicative virus to become corrected or activated. Finally, construction of retrovirus vectors is a highly sophisticated technology requiring a significant amount of expertise. Accordingly, one of the objects of the present invention is to introduce genes into cells which convey immortality without risk of viral replication. Another object of the present invention is to provide a technique for immortalizing B-lymphoid cells using only the DNA necessary for immortalization. Still another object of the present invention is to provide stable, monoclonal antibody-producing cell lines that have been immortalized with an oncogene. Other objects, features, and advantages of the present invention will become apparent from the description of the invention which follows. Brief Description of the Invention In accordance with the foregoing objects, the present invention provides a method for immortalizing lymphoid cells by electrically inserting immortalizing DNA into a target cell. This method comprises the steps of placing the lymphoid cells between two electrodes, adding deoxyribonucleic acid segments containing an oncogene to the cells, and immortalizing at least some of the cells by applying an electric potential to the electrodes to create an electric field of from about 1 to about 40 kV/cm in a pulse of about 0.1 nanoseconds to about 10 milliseconds, to introduce the deoxyribonucleic acid into at least some of the cells. Usually from 2 to 7 pulses are used, and preferably 3 to 5 pulses, having a length of about 5 to about 50 μs and a potential of about 2 to about 10 kV/cm. We have demonstrated that the c-myc gene (of human origin) , when introduced into human B-lymphoid cells either in a plasmid or as a free, linear DNA chain, can stably integrate and immortalize a high proportion of those cells. Although researchers in the field have long recognized that oncogenes have some role in the transformation of some immortalized cells, it has not been previously recognized ,or demonstrated that a single exogenous cellular oncogene can thus transform a B-lymphoid cell. This invention, then, results from the discovery that this single gene can indeed transform B-lymphoid cells, and from the discovery of a simplified, highly-effective method for introducing those genes. In accordance with yet another aspect of the present invention, immortalized cells produced according to the foregoing methods are provided. This invention also includes human lymphoid cells having an exogenous cellular oncogene (preferably human c-myc) incorporated into the genome thereof to immortalize the cell. Those immortalized cells are preferably in the form of individual cell lines capable of producing monoclonal antibody. Detailed Description of the Preferred Embodiments As used in this specification and claims: The term "DNA" or "DNA polymer" shall mean a polymer of deoxyribonucleic acids. The term "naked DNA" shall mean DNA that is not contained in an organism or structure such as a cell, a virus, or a vesicle. Naked DNA is usually free floating DNA in a solution or suspension. The term "exogenous DNA" when used in reference to a cell shall mean DNA from a source external of that cell or of the progenitors thereof. The term "cellular" (or the abbreviation "c") when used to modify "oncogene" shall mean an oncogene taken from a cellular (as opposed to a viral) source. The term "immortal" or "immortalized", when used to describe a cell, shall mean that the cell has acquired the ability to grow indefinitely in vitro without exogenous growth factors. Immortalization requires stable growth for a period of at least several months. Mere production of foci does not demonstrate immortalization. Such immortal cells are usually grown in suspension in vitro. The oncogenes used in the present invention are isolated from mammalian cells, preferably human cells. The ability of any particular oncogene to immortalize lymphoid cells when incorporated into the genome of those cells using the techniques of the present invention can be readily determined by cloning the gene using standard techniques, inserting the cloned gene into a lymphoid cell using the electrical technique described herein, culturing the lymphoid cells so treated, and testing the resulting cell lines for stable growth and protein production. The preferred immortalizing oncogene for use in the present invention is the c-myc oncogene. Procedures have been described for the isolation of the human c-myc oncogene (Dalla-Favera, R. Et al., (Nature 299:61-63 (1982); Dalla-Favera, R. , Proc. Nat'l Acad. Sci. U.S.A. 79:6497-6501 (1982); Taub, R. et al. Proc Nat'l Acad. Sci. U.S.A. 79:7837-7841 (1982); Erickson, et al. Proc Nat'l Acad. Sci. U.S.A. 80 ; 7581-7585 (1983). The human c-myc gene has been mapped to chromosome #8 and restriction enzyme fragments compared to mouse (Dalla Favera, R. et al Proc Nat'l Acad. Sci. U.S.A. 79:7824-7827 (1982)) and the nucleotide sequence and restriction enzyme digestion and fragment analysis described and compared to having c-myc and v-myc (Watson, D.K. et al Proc Nat' 1 Acad. Sci. U.S.A. 80:3642-3645 (1983)). Probes for the isolation and analysis of the human c-myc gene have been described (Lautenberger, J.A. et al Proc Nat'l Acad. Sci. U.S.A. 78:1518-1522 (1981); Dalla- Favera, R. et al. Nature 299:61-63 (1982); Dalla-Favera, R. et al Proc Nat'l Acad. Sci. U.S.A. 79:7824-7827 (1982); Taub, R. et al Proc Nat'l Acad. Sci. U.S.A. 79:7837-7841 (1982); Winman, K.G. et al Proc Nat'l Acad. Sci. U.S.A. 81:6789-6802 (1984)) or are available commercially as Pmyc.1-TM (Oncor, P.O. Box 870, Gaithersburg, MD. 20877). The particular technique used to clone the isolated oncogene is not critical. The human c-myc gene is isolated by the knowledge that high molecular weight human genomic DNA prepared by cell lysis, proteinase K digestion, extraction with phenol, and ethanol precipitation (Levis, R. and S. Penman J. Molec. Biol. 121:219-239 (1978); Wahl, G.M. et al. Proc Nat'l Acad. Sci. U.S.A. 76:3683-3687 (1979)) when digested to completion with the combination of restriction enzymes Eco R1 and Hind III provides an 8.2 Kb fragment containing the complete c-myc gene with three exons (Watson, D.K. et al. Proc Nat'l Acad. Sci. U.S.A. 80:3642-3645 (1983). The gene is preferably isolated from the human promyelocytic leukemia line (Collins, S.J. et al. Nature 270:347-349 (1977)) which contains 16 to 32 copies of c-myc (Dalla- Favera, R. Nature 299:61-63 (1982) but can also be isolated from the human cell veins and tissues which contain the normal and complete c-myc gene (i.e., Taub, R. et al. Proc Nat'l Acad. Sci. U.S.A. 79:7837-7841 1982). The 8.2 Kb EcoR1/Hind III fragment is isolated from the majority of the DNA by adaptation of standard molecular cloning procedures (Maniatis, T. et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1982) and cloned into pUC8 or pBR322 plasmids using Escherichia coli DH1. The restriction enzyme digest is separated by low melting agarose gel electrophoresis. The 8.2Kb area is cut out, heated to 65° to melt agarose, and the DNA fragments extracted with phenol and recovered by ethanol precipitation. The EcoR1/Hind III fragments are identified by using conventional 0.8% agarose gel electrophoresis for separation and Southern blot transfer (Southern E.M. J. Mol Biol. 98:503-517 (1975)) using acid pretreatment (Wahl, G.M. et al. Proc Nat'l Acad. Sci. U.S.A. 76:3683- 3687 (1979)) and hybridization to nick-translated (Ridby, P. . J. Mol. Biol. 113:237-251 (1977)) myc-gene probe (Oncor, Gaithersburg, M.D.). Optimum stringency conditions (hybridization temperature and formamide concentrations) for reaction of this probe against the 8.2 Kb fragment area of the EcoR1/Hind III are developed according to manufacturers suggestions. The 8.2Kb EcoR1/Hind III restriction fragments are ligated into plasmids pUC8 or pBR322 by cutting the parent plasmid vector with EcoR1 and Hind III restriction enzymes and carrying out the ligation reaction with an excess of the 8.2 Kb fragment (i.e., 10μg/2μ of plasmid) and transforming Escherichia coli DH1 (Hanahan, D. J. Mol. Biol. 166:557-580 (1983)) and selecting for ampicillin resistance. The DH1 host is plated at 100,000 cells/20 cm plate and viable colonies are replicated to filter paper (Gergen, J.P. Nucleic Acid Research 7:2115-2135 (1979)) and hybridized against the myc gene probe. The positive areas are scraped into broth cultures and recultured on a picillin containing plates to give spacing. The clones are arrayed and rescreened against the myc gene probe and single colonies are isolated. Alternatively, the 8.2 Kb fragments from the EcoR1/Hind III areas of soft agarose electrophoresis gels can be cloned before plasmid construction by using coliphage lambda charon vector 28 (Rimm, D.L. et al. Gene 12:301-309 (1980)). The appropriate colonies carrying the plasmids containing the 8.2 Kb c-myc gene insert are identified by Southern blot analysis against the myc gene probe of gel electrophoresed samples of EcoR1/Hind III digested plasmid minipreps. The identity of the c-myc gene is established by diagnostic restriction fragment analysis (Watson, D.K. Proc Nat'l Acad. Sci. U.S.A. 80:3642-3645 (1983)); Dalla-Favera, R. Proc Nat'l Acad. Sci. U.S.A. 79:6497-6501 (1982)). Plasmid DNA containing the gene or gene fragments to be used in the electroporation method are prepared by mass culture of the appropriate DH1 stains in ampicillin containing broth, and chloramphenicol amplification of the culture, lysozy e-Triton X-100 lysis of cells (Katz, L.D. et al. J. Bacteriol. 114:577-591 (1973)) and cesium chloride-ethidium bromide equilibrium centrifugation (Radloff, R.W. et al. Proc Nat'l Acad. Sci U.S.A. 57:1514- 1521 (1967)). The isolated naked DNA containing the desired oncogene may be inserted into the lymphoid cell in the plasmid vector. However, transformation and immortalization is more effective when the oncogene is inserted as a linear segment of naked DNA. Appropriate restriction enzymes may be used to linearize and isolate the cloned gene from the plasmid. The c-myc gene may be isolated from pBR322 as an 8.2 Kb segment using Hind III and EcoR1. Cells that can be immortalized using the techniques of the present invention include lymphoid cells, preferably of human origin. Preferred cells are human B-lymphocytes that produce a desired protein or polypeptide. Such proteins and polypeptides include antibodies to particular antigens, T-cell growth factor, and B-cell growth factor. In the process of the present invention, a cell suspension containing the desired lymphoid cells is prepared. A relatively nonconducting suspension, such as about 0.3M mannitol, sorbitol, glucose, sucrose, or histidine is preferred. The naked DNA segments containing the oncogene are added to the suspension, and the suspension is placed between two electrodes. Good results are obtained when the electrodes are spaced 0.3-1.0 cm apart. A standard electrofusion chamber containing such electrodes, such as the electrofusion chamber manufactured by Maxwell Manufacturing Co. of San Diego, can be used. Insertion of the DNA into the lymphoid cells is accomplished by delivering at least 1 and preferably 2 to 7 electric pulses to the cell suspension containing the naked oncogene. The use of from 3 to 5 pulses is 2" preferred. The pulse length may be from about 0.1 ns to about 10 ms. Usually the pulse length is about five to about fifty μs. Any suitable electric source capable of delivering an electric potential to the electrodes of from about 1 to " about 40 kV/cm may be used. Usually a potential of about 2 to about 10 kV/cm is used. The pulse may be generated by any conventional technique, such as by use of a pulse transformer and/or capacitor discharge. Several commercially available pulse generators in standard 3 electrofusion equipment, such as that developed by Maxwell Manufacturing Co. of San Diego, can provide suitable electric pulses. Another suitable pulse generator is Hewlett Packard Model 214B. It has been hypothesized that application of an electric pulse to a cell renders the cell membrane transiently permeable. See Zimmerman and Greyson, "Electric Field-Induced Cell Fusion", Bio Techniques, September/October 1983: 118. Following the electric pulse treatment, the cell suspension is allowed to sit for about 10 to 15 minutes at a low temperature, e.g., 4°C or at room temperature. It is believed that the rest period is required for a return of the electroporated cell membrane to a normal condition, during which time the cell membrane remains permeable to the naked exogenous DNA. The cell suspension is then diluted and seeded into an appropriate culture medium. After incubation at 37°C for a number of days, growth of the immortalized cells will be observed. Single colony isolates may then be obtained and the particular cells producing the desired protein may be identified and cultured using standard techniques. This technique is particularly important in obtaining monoclonal antibody-producing cell lines of human origin. Because of ethical constraints, conventional techniques for obtaining cell samples from animals in which a comparatively large numbers of lymphoid cells produce the desired antibody cannot be used for obtaining human cells. Therefore, a technique that exhibits high transformation efficiency is particularly valuable, because the concentration of the desired cell in the human cell suspension is extremely low. Unlike previously created cells, the cells produced according to the present invention have an exogenous cellular oncogene incorporated into the genome thereof, making the cell immortal. Such immortalized human lymphoid cells are considered a part of the present invention. The antibody-producing cells resulting from this invention differ from those resulting from previous methods. No known transforming virus or viral genome has been introduced. Oncogenes are thought to provide natural signals which are used during differentiation and maturation of cells. Normally, their expression is controlled by the location or proximity to normal control regions or signals in the chromosome. In the transformed cells of the present invention, it is believed that the oncogenes have simply integrated into a location where expression is not prevented by normal cellular control mechanisms. There have been no alterations in the chromosomal load; i.e., no additional chromosomes have been introduced. EXAMPLE A fresh tonsil was dispersed into cell suspension by passing through a garlic press. The resulting tonsilar cell suspension in Hank's buffered salt solution was stored at 4°C until use. A recombinant pBR322 plasmid containing the human c-myc gene isolated from the promyelocytic leukemia cell line HL-60 was obtained as described in D. Favera, et al., "One Gene Amplification in Promyelocytic Leukemia Cell in HL-60 and Primary Leukaemic Cells of the Same Patient" , Nature 299: 61-63 (1982) and D. Favera, et al., "Cloning and Characterization of Different Human Sequences Related to the One Gene (c-myc) of Avian Myelocytomatosis Virus (MC29)", Proc. Nat'l. Acad. Sci. U.S.A. 79: 6497-6501 (1982). The plasmid was transfected and ampicillin amplified in escherichia coli by standard procedures. See, e.g., F. Bolivar, et al. , Gene 2: 95-113 (1977); Morrow, et al., Proc. Nat'l'. Acad. Sci. U.S.A. 71: 1743-1747 (1974); Grunstein & Hugress, Proc. Nat'l'. Acad. Sci. U.S.A. 72: 3961-3965 (1975); and Bernard & Helinski, Genetic Engineering Principles and Methods 2 (1980). The resulting plasmids were purified and either used as is or as the c-myc gene (8.2 Kb insert) following Hind III/EcoR1 digestion and further purification. Tonsil cells prepared as above (approximately 50% B- lymphocytes) were centrifuged at 500 xG for five inutes. Hanks buffer was removed by aspiration and the cell pellet was resuspended in cold, sterile 0.3M mannitol, pH 7.2, to a final cell density of 2-5 x 10° cells/ml. The c-myc gene or plasmid containing the gene [approximately 1 mg/ml in 10mM Tris-HCl, 0.5mM sodium EDTA, pH 7.4] was added to the cell suspension. Generally 10 μl was added, which represents 10^-** to 10^ genes per ml. The suspension was gently mixed and transferred to a multiple pre-cooled stainless steel electrofusion chamber made by Maxwell Corporation of San Diego. The lymphocytes were rendered permeable to the c-myc genes or plasmids by four electric pulses creating a field strength between the electrodes of of 4 kV/cm to 7 kV/cm. The pulse lengths were 5 to 50 μ sec. The suspension was gently mixed for 10 to 20 seconds between pulses. The pulsed cell suspension was allowed to sit on ice at about 4°C for approximately 15 minutes. The suspension was then diluted 1:10 to 1:15 with RPMI-1640 medium containing 10 percent fetal calf serum (FCS) . The resulting diluted suspension W s carefully centrifuged and resuspended in the RPMI-10% FCS to 5 x 10 5 to 10 6 cells/ml. One ml aliquots then were seeded into each of the wells of a 24-well culture dish. The cultures were incubated at 37°C under a five percent CO2 atmosphere until growth was apparent; generally 7 to 10 days. The cultures were expanded, were media tested for presence of immunoglobulin, and permanent collections were established by freezing at -70°C. Single colony isolates were obtained by standard soft agar or limiting- dilution cloning techniques. The clones were expanded and characterized with regard to growth properties, antibody production and type, and restriction fragment patterns. That growth of the antibody-producing B-lymphocyte is dependent on c-myc insertion was demonstrated by our failure to observe growth in similar experiments using plasmids not containing the c-myc gene, using genes unrelated to c-myc, and using lymphocytes subjected to electric pulses in the absence of any exogenous source of nucleic acid. The immortalized monoclonal-antibody producing cell lines have exhibited stable growth and antibody production for over four months and continue to exhibit such growth and antibody production. Although the present invention has been described in terms of certain preferred embodiments, it will be understood that the scope of the present invention is to be defined by the following claims, and reasonable equivalents thereof.

Claims: WHAT IS CLAIMED: 1. A method for immortalizing human lymphoid cells, comprising the steps of: placing a human lymphoid cells between two electrodes; adding naked DNA polymer segments containing a cellular oncogene to said cells; and immortalizing at least some of said cells by applying an electric potential to said electrodes to introduce said oncogene into at least some of said cells. 2. A method as claimed in Claim 1, wherein said lymphoid cells comprise B-lymphocyte cells producing antibody to a predetermined antigen and wherein said cells are placed between said electrodes in the form of a suspension of said cells to which said potential is applied in the form of at least one high voltage electric pulse. 3. A method as claimed in Claim 1 or 2, wherein said step of applying said electric potential comprises creating an electric field of from about 1 to about 40 kV/cm in a pulse having a pulse width of about 0.1 nanosecond to 10 milliseconds. 4. A method as claimed in Claim _, wherein from 2 to 7 pulses are delivered to said cells, said pulses having a pulse width of about 5 to 50 us and a potential of about 2 to 10 kV/cm. 5. A mehtod as claimed in any of Claims 1 to 4, wherein the oncogene is a c-myc gene. 6. A method as claimed in any of Claims 1 to 5, wherein the oncogene is a human oncogene. 7. A method as claimed in any preceding Claim, wherein the DNA polymer segments are plasmids. 8. A method as claimed in any of Claims 1 to 6, wherein the DNA polymer segments are linear. 9. A method as claimed in Claim 2, further including the steps of: culturing the immortalized cells in vitro; and isolating individual cell lines producing monoclonal antibody to the antigen. 10. An immortalized human B-lymphocyte cell prepared by the method of any of Claims 1 to 9. 11. A human lymphoid cell having an exogenous cellular oncogene incorporated into the genome thereof to immortalized said cell. 12. The cell of Claim 14, in which the oncogene is c-myc. 13. The cell of Claim 14, in which the oncogene is human c-myc and the cell is a B-lymphocyte. 14. The cell of Claim 14, in the form of a culture capable of producing monoclonal antibody.

Inventors: LUNDAK ROBERT L, LUNDAK, ROBERT, L.
Applicants: TECHNICLONE RESEARCH PARTNERS, TECHNICLONE RESEARCH PARTNERS I
Application year: 1985
Main classification: C12N15
IPC codes: C12N5, C12N13, C12N15, C12R1