Proc Natl. Acad. Sci. USA
`Vol. 80, pp. 825-829, February 1983
`Immunology
`
`Immunoglobulin gene expression in transformed lymphoid cells
`(gpt/transformation)
`
`VERNON T. 01*, Srnzrurs L. Moruusom‘, LEONARD A. HERZENBERC*, AND PAUL Brtnci
`ent of Microbiology and the Cancer
`De
`ents of ‘Genetics and iliiochemistry, Stanford University
`partm
`School of Medicine, Stanford, Califomia 94305: and 3De
`Center, Institute ofCancer Research, College of Physicians and Surgeons of Columbia University, New Yorlt, New York 1
`
`Contributed by Leonard A. I-Ierzenberg, October 15, 1982
`
`Myeloma, hybridoma, and thymoma cell lines
`ABSTRACT
`have been successfully transfected for the Escherichia cola’ xan-
`thine-guanine phosphorihosyltransferase gene (gpt) by using the
`plasmid vector pSV2-gpt. The transformed cells synthesize the
`bacterial enzyme 5-phospho-at-D-ribose-l-diphosphatezxanthine
`phosphoribosyltransferase (XGPRT; EC 2.4.2.22) and have been
`maintained in selective medium for over 4 months. Lymphoid cell
`lines expressing a K immunoglobulin light chain were obtained by
`transfecting cells with pSV2-gpt containing a rearranged K light
`chain genomic segment from the S107 myeloma cell line. The $107
`light chain is synthesized in gpt-transformed ]558L myeloma cells
`and is identical to the light chain synthesized by the S107 myeloma
`cell line, as judged by immunoprecipitation and two-dimensional
`gel electrophoresis. Furthermore, this light chain is synthesized
`and secreted as part ofan intact antibody molecule by transfonned
`hybridoma cells that normally secrete an IgGl (y,K) antibody
`molecule. No light chain synthesis was detected in a similarly
`transformed rat myeloma or a mouse thymoma line.
`
`Techniques to introduce novel genes into eukaryotic cells pro
`vide a powerful tool to study mechanisms ofgene regulation and
`expression. Most studies on eukaryotic gene expression have
`been conducted in heterologous host cells—i.e., genes have
`been transfected into cell types (particularly human HeLa and
`mouse L cells) that normally do not express the gene ofinterest
`(1-3). Though a great deal has been learned about eukaryotic
`regulator sequences with these gene transfer experiments, it
`would be preferable to transfer genes encoding proteins ex-
`pressed during diflerentiation back into the cell type that nor-
`mally expresses the genes ofinterest. The appropriate cell type
`provides protein modification systems, such as glycosyltrans-
`ferases, necessary to make fully biological functional products.
`In addition, the appropriate cell type may be used to study tis-
`sue-specific regulation of gene expression.
`To undertake studies of (i) the regulation and expression of
`immunoglobulin genes, (ii) the biosynthesis, chain-assembly,
`and secretion of immunoglobulin heavy and light chains, and
`(iii) structure—function correlates of antibody molecules, we
`have explored techniques for transfection of lymphoid cells us-
`ing the pSV2-gpt vector (4, 5). This DNA can express the Eco
`gpt gene encoding xanthine-guanine phosphoribosyltransferase
`(XGPRT; 5-phospho-at-D-ribose-l-diphosphatezxanthine phos-
`phoribosyltransferase, EC 2.4.2.22). Cells synthesizing XCPRT
`can be grown with xanthine as the sole precursor of guanine
`nucleotide formation (4, 5). Successfully transformed cells can
`be isolated by their ability to grow in medium containing xan-
`thine and mycophenolic acid, an inhibitor of guanine nucleotide
`synthesis; if the transfomred cell line is hypoxanthine phos-
`phoribosyltransferase-negative (HPRT'; IMP pyrophosphate
`phosphoribosyltransferase, EC 2.4.2.8), transformants can he
`
`The publication costs ofthis article were defrayed in part by page charge
`payment. This article must therefore be hereby marked “advertise-
`ment” in accordance with 18 U. S. C. §l734 solely to indicate this fact.
`
`825
`
`selected in hypoxanthine/aminopterin/thymidine (HAT) me-
`dium (6). In the_present experiments both calcium phosphate
`precipitation (7, 8) and protoplast fusion (9) techniques have
`been used to transfect cells.
`pSV2-gpt containing a rearranged K light chain gene (10) was
`used to transform several cultured lymphoid cell lines. Among
`the gpt transformants were clones that produce a new immu-
`noglobulin light chain. The light chain produced by these trans-
`formed cell lines appears to be identical to the light chain syn-
`thesized by the myeloma cell from which the rearranged gene
`was isolated. Furthermore, in transfonned hybridoma cells,
`this light chain is assembled with an immunoglobulin heavy
`chain and secreted as a complete antibody molecule.
`
`MATERIALS AND METHODS
`
`Cell Lines. ]558L is a spontaneous heavy chain-loss-variant
`myeloma cell line obtained from the ]558 cell line [a,/\; anti-al-
`3 dextr-an (ll)] that synthesizes and secretes a A light chain. Y3-
`Agl.2.3 is a HPRTrat myeloma cell line originally described
`by Galfre et al (12) that synthesizes and secretes a rat K light
`chain. 27-44 is a HPBT‘ mouse IgC1 anti-dansyl hybridoma cell
`line (13); and BWSI47 is a HPRT‘, ouabain' AKR thymoma
`originally described by Hyman and Stallings (14). Cell lines
`were maintained in either 10% newborn calf serum in Dul-
`becco’s modified minimal essential medium (DME medium) or
`10% fetal calf serum in alpha modified minimal essential me-
`dium.
`Recombinant DNA Vectors. The plasmid vector pSV2-gpt
`has been described (4, 5). Fig. 1 shows a partial restriction en-
`zyme map ofthis vector. A second vector, which is derived from
`pSV2-gpt, but contains the herpes simplex thymidine kinase
`promoter inserted 5’ of the gpt gene, was constructed by ].—F.
`Nicolas (unpublished data). pSV2-S107 was constructed by in-
`serting a BamHI fragment containing the entire rearranged
`phosphocholine-specific K chain gene from the S107 myeloma
`cell line (10) into the unique BamHI site in pSV2-gpt. The light
`chain gene is oriented so that the direction of transcription is
`opposite to the gpt gene (Fig. 1). The genomic rearranged S107
`K light chain DNA was a gift from M. Scharff.
`Transfection by Protoplast Fusion. Protoplasts were pre-
`pared essentially as described by Sandri-Coldin et al. (9). Each-
`erichia coli K-12 strain HB 101, containing the appropriate plas-
`mid, was grown at 37°C in Luria broth containing 1% glucose
`to an absorbance at 600 nm of 0.6—0.8. Chloramphenicol was
`added to 125 p.g/ml, the culture was incubated at 37°C for 12-
`16 hr to amplify the plasmid copy number, and the cells were
`harvested by centrifugation. For every 25 ml of culture, 1.25
`ml of chilled 20% sucrose/0.05 M Tr-is'HCl (pH 8) was added;
`
`Abbreviations: XCPRT, xanthine-guanine phosphoribosyltransferase;
`HPRT, hypoxanthine phosphoribosyltransferase; HAT, hypoxanthine/
`aminopterin/thymidine; DME medium, Dulbeccds modified minimal
`essential medium.
`
`Sanofi/Regeneron Ex. 1031, pg 915
`
`Mylan Ex. 1031, pg 915
`
`

`
`826
`
`Immunology: Oi et al.
`
`Proc. Natl Acad. Sci. USA 80 (1983)
`
`pBR322 mi
`
`Ampn
`
`
`
`
`
`FIG. 1. Structure of the vectors
`used for lymphoid cell transforma-
`tions. The diagram of the parental
`pSV2-gpt plasmid vector was taken
`from Mulligan and Berg (4, 5): pBR322
`DNA is represented by the solid black
`lines and the pla.smid’s DNA replica-
`tion origin and B-lactamase gene are
`indicated; the git gene sequence is
`represented by the hatched segments;
`simian virus 40 (SV40) sequences are
`the stippled segments. The SV40 ori-
`gin of DNA replication (ori) and early
`promoter are located 5' of the gpt se-
`quences. pSV2-gptTKpr has an inser-
`tion of 250 base pairs, containing the
`herpes simplex thymidine kinase pro-
`moter, between the gpt gene and the
`SV40 early promoter (unpublished
`data). pSV2-S107 has a 7-kilobase
`Baml-II fragment, containing the en-
`tire genomic S107 light chain gene,
`inserted into the unique BamHI site
`of pSV2-gpt. This rearranged light
`chain gene is oriented in the opposite
`direction togpt and contains the leader,
`V, and x constant region exons as well
`as flanking 5’ and 3’ sequences.
`
`the bacteria were suspended and 0.25 ml of lysozyme [a freshly
`prepared solution of 5 mg/ml in 0.25 M Tris-HCI (pH 8)] was
`added. After 5 min ofincubation on ice, 0.5 ml of 0.25 M EDTA
`(pH 8) was added and incubation on ice was continued for an
`additional 5 min. After addition of 0.5 ml of 0.05 M Tris'HCl
`(pH 8), the bacteria were transferred to a 37°C water bath and
`were incubated for 10 min. At this time examination of the bac-
`teria with a phase-contrast microscope showed that the vast
`majority had been converted to protoplasts. The bacteria were
`diluted with 10 ml of DME medium containing 10% sucrose
`and 10 mM MgCl2 that was warmed to 87°C. After further in-
`cubation for 10 min at room temperature the protoplasts were
`ready for fusion.
`Fusion of protoplasts with suspension cells was effected with
`a procedure normally used in the production of hybridomas
`(15). Cell lines were grown to a density of 0.3-1 X 106 cells per
`ml in DME medium supplemented with 10% newborn calf
`serum. Five milliliters of the protoplast suspension was added
`to 2 X 106 cells in growth medium. The mixture was centrifuged
`for 5 min at room temperature at approximately 500 X g. The
`supemate was aspirated and the pellet was resuspended gently
`in 2 ml of a polyethylene glycol solution [50 g of polyethylene
`glycol 1,500 (BDH) in 50 ml of DME medium] adjusted to pH
`8 with C02. After 3 min of centrifugation at 500 X g the poly-
`ethylene glycol was diluted with 7 ml of DME medium while
`resuspending the pellet. After 5 min of centrifugation at 500
`X g, the supemate was removed carefully and the cells were
`resuspended in DME medium containing 10% newborn calf
`serum and garamycin at 100 pg/ml and were plated either in
`96-well or 24-well plates. After 48 hr, cells were diluted with
`an equal volume of DME medium containing xanthine at
`;Lg/ml, hypoxanthine at 15 pg/ml, mycophenolic acid at 6 ;1g/
`ml, and 10% newborn calf serum. Every several days, as re-
`quired, spent medium was aspirated carefully and was replaced
`with fresh medium containing the same supplements. Colonies
`of transfonnants were visible by 10 days. Transformants were
`maintained in selective medium.
`Transfection by Calcium Phosphate Precipitation. Lym-
`phoid cell lines grown in suspension were transfected by cal-
`cium phosphate precipitation as described by Chu and Sharp
`(7). Ten times concentrated HeBS buffer was stored at -20°C
`
`until used, whereupon it was diluted to two times concentrated
`and adjusted to pH 7.05. Plasmid DNA (80 pg/ml) was made
`up in 125 mM CaCl2 which was stored as a 2 M stock solution
`at -20°C. DNA-mlcium phosphate precipitates were formed
`by dropwise addition of the DNA into the HeBS solution. The
`precipitate formed in 30 min at room temperature. The final
`DNA concentration was 40 pg/ml.
`Cells were washed once in serum-free medium and were
`suspended directly in the DNA-calcium phosphate precipitate
`(106 cells per 20 pg of DNA per 0.5 ml). This suspension was
`incubated at 37°C for 30 min and then was diluted 1:10 in
`serum-containing medium. The cells were plated either into 24-
`well plates (2 X 105 cells per well) or 96-well plates (2 X 10‘
`cells per well). Transfection of Y3 cells was done as described
`by Graham and Van der Eb (8) for adherent cell lines. The DNA-
`calcium phosphate precipitate was put directly onto the cell
`monolayer. After 30 min at 37°C, serum-containing medium
`was added. After 24 hr, half of the medium volume from each
`culture was removed and was replaced with fresh medium. On
`days 3, 4, 5, 8, 11, and 14, half the medium volume was re-
`moved and HAT medium was added. Transformed colonies
`were visible between 10 and 21 days.
`Immunoprecipitations and Cel Electrophoresis. Immuno-
`precipitations were done with [”S]methionine-labeled cell ly-
`sates and supemates. Biosynthetic labeling procedures have
`been described (16). Rabbit anti-mouse light chains, rabbit anti-
`mouse K light chains, rabbit anti-mouse immunoglobulin, and
`a hybridoma anti-mouse IgGl allotype antibody were used for
`immunoprecipitations. Staphylococcus aureus, Cowan strain 1
`(IgCsorb; Enzyme Center, Boston) was used to coprecipitate
`the antigen—antibody complexes (16).
`One-dimensional NaDodSO4/polyacrylamide slab electro-
`phoresis and two-dimensional nonequilibrium gradient gel
`electrophoresis were done as described (17). Autoradiography
`ofpolyacrylamide gels was with preflashed XAR-5 film and fluo-
`rography by using sodium salicylate (18).
`
`RESULTS
`
`Transfection Frequencies. The frequency at which stable
`transformed lymphoid cell lines were generated was influenced
`
`Sanofi/Regeneron Ex. 1031, pg 916
`
`Mylan Ex. 1031, pg 916
`
`

`
`Immunology: Oi et at
`
`Proc. Natl. Acad. Sci. USA so (1983)
`
`827
`
`Table 2. Transformation of lymphoid cell lin with the pSV2-
`gptvectors by using calcium phosphate precipitation
`Cell line
`27-44
`
`BW5147
`
`Vector
`
`Y3
`
`pSV2-gpt
`pSV2-g‘ptTKpt‘
`pSV2-S107
`
`3/48‘
`19/48*
`47/48*
`
`10/192*
`10/192*
`43/288*
`
`1/192*
`1/192*
`0/192*
`
`Results are from three experiments with the Y3-cell line and two
`experiments with 27-44»and BW5147 cell lines.
`’ Cells were plated in 24-well culture dishes at 2 X 10‘ cells per well."
`7 Cells were plated-in 9l‘rwell culture dishes at 4 X 10‘ cells per well.
`
`Ofthe four cell lines stably transformed with pSV2-S 107, the
`]558L cell line synthesized, but didnot secrete, the S 107 K light
`chain. However, this cell line was not expected to secrete the
`newly made light chains because heavy chain-loss-variants of
`the S107 myeloma cell line also do not secrete endogenous light
`chains (M. Scharlf, personal communication). Transforrnants of
`the 27-44 hybridoma cell line synthesized and secreted the S107
`light chain. Moreover, the S107 light chain was assembled into
`tetrameric H,L2 immunoglobulin molecules with the endoge-
`nous yl heavy chainand was secreted.- Twelve independently
`transformed Y3 and seven BW5147 cell lines did not produce
`detectable amounts ‘of the. S107 light chain, as judged by im-
`munoprecipitation andgel analyses. XCPRT analyses verified
`that these- cells were, indeed, transformants.
`Autoradiograms of two-dimensional polyacrylamide gels
`showing the apparent M,‘ and charge of the light chains pro-
`duced by ]558L and 27-44 transformants are shown in Figs. 3
`and 4. The two-dimensional gel pattern. of the S107 light chain .
`synthesized by $107 myeloma cells is included to show that the
`transformed cell lines produced a light chain that is identical
`in apparent M, and charge. The two-dimensional gel patterns
`also show that the leader polypeptide was removed in trans-
`formed cell lines that expressed the light chain. This indicates
`that proper transcription, mRNA, and protein processing occur
`in the transfonnants. Transcription of the S107 light chain gene
`probably occurs from its own promoter, because the light chain
`gene is oriented opposite to the direction of the SV40 early pro-
`moter (see Fig. 1).
`The antibodies secreted by 27-44 transformants were im-
`munoprecipitated with both hybridoma anti-IgCl allotypic an-
`tibody and rabbit anti-mouse light chain antisera. Both reagents
`precipitated the S107 light chain (data not shown). Sequential
`precipitation, first with the hybridoma anti-IgCl antibody and
`
`1
`
`2
`
`FIG. 2. XGPRT and I-[PRT production in transformed lymphoid
`cell lines. Enzyme analyses were done as described by Mulligan and
`Berg (4, 5). Lanes: 1 and 2, electrophoretic mobility of mammalian
`HPRT; 3-5, J558L cell transformants; and 6 and 7, transformants of
`the 27-44 cell line. Because 27-44~is a HPRT.‘ cell line, only XGPRT
`is present.
`
`Sanofi/Regeneron Ex. 1031, pg 917
`
`Mylan Ex. 1031, pg 917
`
`by every parameter tested. Different cell lines and different
`vectors produced different transformation frequencies. More-
`over, the two DNA delivery procedures, protoplast firsion and
`calcium phosphate precipitation, yielded different
`transfor-
`mation frequencies. Tables land 2 summarize the results by"
`using protoplast fusion and calcium phosphate precipitation,
`respectively.
`Under the present experimental conditions, BW5l47 ap-
`pears tobe the least competent recipient ofthe cell lines tested,
`having a transformation frequency of approximately 10'“. Y3
`and 27-44 yielded frequencies in the rangeof0.3 to >5.>< 10’°.
`In- the present experiments, ]558L yielded the highest fre-
`quency with the range of 3 X l0'° to >10". Protoplast firsion
`appears on balance to be a more efficient delivery system than ~
`calcium phosphate precipitation.
`A striking feature of these results is the enhanced transfor- ‘
`mation frequency for gpt obtained with the light chain-contain-
`ing vector, pSV2-S107. This dramatic increase is evident when
`the pSV2-S107 vector was used with the ]558L and Y3 myeloma
`cell lines; transformation with this recombinant was 5- to at least
`10-fold greater than that obtained with the other. vectors. Trans-
`formation of the hybridoma 27-44 ‘cell line was increased only
`about 2-fold with pSV2-S107. The sequence(s) in the pSV2-S107
`insert that is responsible for the enhanced transformation fre-
`quency must yet be mapped. Transformation of the Y3 cell line
`was occasionally greater with pSV2-gpt-'I'l(pr than with pSV2-
`gpt (Table 2). Regardless of which vector was used, BW5l47
`transformants were detected only at ‘very low frequencies. The
`amount of XGPRT activity in cell lines stably transformed by
`the three recombinant plasmids was not significantly different
`(Fig. 2 and data not shown).
`XGPRT Activity. The transformed cell lines expressedvthe
`Eco gpt gene, as measuredby the presence of XCPRT activity
`in the cell lysates. E. colt‘ XGPRT can be distinguished from
`mammalian HPRT activity by its different electrophoretic mo-.
`bility (4, 5). In cells selected for resistance to mycophenolic acid,
`both the cellular HPRT and bacterial XGPBT activities were
`detectable (Fig. 2). Cells lacking their own HPRT activity and
`selected for gpt in HAT medium had only the bacterial enzyme
`activity (Fig. 2).
`Immunoglobulin Light Chain Expression. The organization
`of exons in the S107 genomic light chain gene is shown in Fig.
`1. To produce the S107 light chain protein from this gene, two
`introns must be processed fiom the primary mRNA transcripts
`and the leader polypeptide removed by post-translational cleav-
`age. For secretion of the light chain as part of an intact antibody
`molecule, the newly synthesized light chain must fold and as-
`semble with an immunoglobulin heavy chain to form an H214
`tetramer. This also involves the fonnation of interchain disul-
`fide bonds.
`
`-
`
`Table 1. Transformation of lymphoid cell lina with the
`pSV2-g-pt vectors by usingprotoplast fusion
`Cell line
`
`Vector
`
`J558L
`
`BW5147
`
`0/76‘ 1/48*
`0/80‘ 1/48*
`4/96* 8/48'
`
`pSV2-gpt
`pSV2-gptTKpr
`pSV2-S107
`
`21/288‘ 27/36*
`10/190‘ 24/36*
`186/192‘ 36/367
`149/192:
`Results are from three experiments and are expressed as the number
`of culture wells having stable transformants.
`' Alter pmtoplast fusion cells were plated in 96-well culture dishes at
`10‘ cells per well.
`1 Cells were plated at 10‘ cells per 2.0 ml of culture in 24-well dishes.
`1 Cells were plated at 5 X 103 cells per well in 96-well cultu.re.disbes.
`
`

`
`828
`
`Immunology: Oi et al.
`
`Proc. Natl. Acad. Sci. USA 80 (1983)
`
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`FIG. 3. S107 light chain produced by transformed 27-44 cells. Au-
`toradiograms of twtrdimensional gels of the light and heavy chains
`produced by parental and transformed cell lines are shown. (A) Pa-
`rental 27-44 IgG1 anti-dansyl antibody immunoprecipitated with an
`anti-IgG1-specific hybridoma antibody. Both the yl heavy and x light
`chains can be seen. (B) S107 IgA antibody immunoprecipitated with
`a rabbit anti-IgA antiserum. The a heavy chain was distinguished
`clearly by charge and apparent M, from the )4] heavy chain in A. (C)
`A mixture of the immunoprecipitates of A and B. The two at light
`chains can be seen as distinct spots (indicated by arrows) having
`nearly identical charge but different apparent M, (D) Immunoprecip-
`itate of a transformed 27-44 cell line. Only the 141 heavy chain was
`present, but two light chains can be seen. In this case the amount of
`S107 light chain was considerably lower than in the artificial mixture
`shown in C.
`
`then with the rabbit anti—K antisera, indicated that little, if any,
`free S 107 light chain was secreted by these cells. This shows that
`the S107 light chain is assembled with the 71 heavy chain into
`an intact antibody molecule.
`Different amounts of S107 light chains were produced when
`a number of independent ]558L and 27-44 transformants were
`
`FIG. 4. S107 light chain produced by transformed J558L cells. Au-
`toradiogrsms of two-dimensional gels of light chains immunoprecip-
`itated from cell lysates of J558L transformed with pSV2-S107 DNA
`are shown. Because J558L does not produce a heavy chain, only the
`light chain portions of the two-dimensional gels are shown. (A) A light
`chain produced by the parental J558L cell line. (B) S107 x light chain.
`(C) A mixture of the two light chains. The K and A light chains are
`distinguished on the basis of both charge and apparent M, (D and E)
`Two independently derived J558L cell lines transformed with pSV2-
`S107 DNA. The tranaformant examined in D only appears to produce
`larger quantifies of the S107 1: light chain than the endogenous J558
`A light chain, because the S107 K chain is synthesized and remains in
`the cytoplasm, while the J558 A chain is synthesized and secreted.
`
`compared. Amounts varied from barely detectable to quantities
`equal to endogenous light chain. This variation may be due to
`the chromosomal region where the light chain has integrated.
`It also could result from different copy number ofthe light chain
`gene in different transformants. Quite possibly, mutations or
`deletions of sequences needed for the expression of this gene
`could have occurred during transformation or subsequent to
`integration of the light chain sequence. Further studies are
`needed to detennine the cause of this variation and why light
`chain expression does not occur in Y3 or BWSI47 cell lines
`transfonned with the same light chain gene vector.
`
`DISCUSSION
`
`These experiments show that it is possible to use two methods,
`calcium phosphate precipitation and protoplast fusion, to intro-
`duce genes into lymphoid cells. With pSV2-gpt containing the
`gene for an immunoglobulin light chain (pSV2-S107) both meth-
`ods give rise to transformants that synthesize bacterial XGPRT
`and the murine light chain. Higher transfonnation frequencies
`are seen following protoplast fusion. Indeed, by using protoplast
`fusion and the pSV2-S107 plasmid, transformants can be ob-
`tained at a frequency of greater than 10“. Transformation fre-
`quencies are lower when using the other plasmids or calcium
`phosphate precipitation. Because mycophenolic acid resistance
`or reversion of the HPRT' phenotype do not occur sponta-
`neously in the cell lines used, stable transformation, at even low
`frequencies, can be detected.
`A surprising result is the increased frequency of gpt trans-
`formation when the SIO7 light chain is incorporated into the
`
`Sanofi/Regeneron Ex. 1031, pg 918
`
`Mylan Ex. 1031, pg 918
`
`

`
`Immunology: Oi et al
`
`Proc. Natl Acad. Sci. USA 80 (1983)
`
`829
`
`pSV2-gpt vector. This enhancing effect occurs with both the rat
`and mouse myelomas». A similar increased transformation fre-
`quency has been observed with a bovine papillomavirus vector
`containing the human B-globin region sequences (19). At pres-
`ent, the mechanism for the increased transformation frequency
`in both cases is obscure. Possibly, the chromosomal DNA pro-
`vides an origin of DNA replication, which permits the plasmid
`to replicate within the transformed cell and increases the trans-
`formation frequency. Transcription fi'om the immunoglobulin
`promoter cannot be essential for the increased transformation
`frequencies because deletion of the fragments that are pre-
`sumed to contain the immunoglobulin promoter region does not
`abolish the ‘enhancement of transformation. It also is possible
`that pSV2-S107 is more efficient for transformation because of
`increased XCPRT production;
`this seems unlikely because
`there are no consistent differences in enzyme levels in the stable
`transformants obtained with either vector.
`DNA-mediated gene transfer into lymphoid cells may permit
`a study of the regulation and expression of immunoglobulin
`genes in cells in which they normally are synthesized. It may
`be possible to examine the basis for differential immunoglobulin
`gene expression at different stages of lymphocyte differentia-
`tion. Cell lines in which immunoglobulin synthesis (:31. be in-
`duced (20-22) are suitable hosts to determine if the transduced
`immunoglobulin genes also are responsive to those signals.
`Studies with cells transformed with genetic elements that are
`inducible by steroid hormones demonstrate that transduced
`DNA can respond, if thecell contains the appropriate receptors‘
`
`A question of central importance is what determines the uti-
`lization of various promoters and thus the synthesis of defined
`proteins in certain cell lines. In our experiments light chains
`are elliciently produced in both transformed mouse myeloma
`and hybridoma cell lines. However, light chain production did
`not occur in either a rat myeloma or a mouse thymoma. The
`inability of the immunoglobulin promoter to function in a dif-
`ferent species has been reported by Falkner and Zachau (24).
`The lack of production of mouse immunoglobulin in a rat my-
`eloma is surprising because mouse myelomas have been used
`to fuse to rat myelomas. to produce hybrid cells that synthesize
`both rat and mouse immunoglobulin molecules (25). The pos-
`sibility that the S107 light chain is synthesized but rapidly de-
`yaded in the Y3 myeloma has not been excluded.
`There is evidence that differentiated cell types express im-
`munoglobulin genes to varyinglevels. For example, somatic cell
`hybridization of myelomas yields hybridomas that produce an-
`tibodies, whereas thymomas yield hybrid cells with T-cell phe-
`notypes (26). Furthermore, hybridization of myelomas with
`non-B cells results in cessation of immunoglobulin production
`(26, 27).. The lack of light chain expression in the transformed
`thymoma may reflect tissue-specific gene regulation. It is im-
`portant to determine if immunoglobulin gene expression in the
`nonexpressing mouse thymoma and rat myeloma cell lines is
`regulated at the level of transcription, RNA processing, trans-
`lation, or rapid protein turnover.
`The study of the structure and function of the immunoglob-
`ulin molecule has been of great interest, both because of the
`ability of immunoglobulin to react with a diverse family of li-
`gands and also because of the biologic importance of antibody
`molecules. Initially, the study of immunoglobulins was limited
`to the study of heterogeneous serum pools after immunization.
`The advent of myelomas, and more recently hybridomas, has
`permitted the study ofhomogeneous populations ofantibodies.
`DNA-mediated transfection and immunoglobulin gene expres-
`sion is an important tool to permit the study of immunoglobulin
`
`molecules. By using this technique, it should be possible to
`study the function of both novel chain combinations and novel
`chain structures. In vitro site-specific mutagenesis techniques
`can be used to construct specific mutations in immunoglobulin
`genes that can be expressed after transfection. Because signif-
`icant quantities of immunoglobulin are produced in the trans-
`fonnants, sufficient quantities of protein necessary for detailed
`analyses should be obtained.
`Note Added in Proof. After this paper was submitted for. publication,
`we leamed that Douglas Rice and David Baltimore have reported sim-
`ilar results with a different K light chain gene and different lymphoid
`cell recipients (28).
`We thank Dr. P. Jones for assistance with two-dimensional gel elec-
`trophoresis. This work was supported in part by National Institutes of
`Health Grants CM-13235, CA-15513 (P.B.), AI-08917 (L.A.H.), CA-
`16858 (S. L. M.), CA-22736 (S. L. M.), and CA-13696 to The Cancer Cen-
`ter of Columbia University and by a grant from the Becton Dickinson
`FACS Systems (L.A.H.). S.L.M.
`is a recipient of Research Career
`Development Award AI—00408.
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`Sanofi/Regeneron Ex. 1031, pg 919
`
`Mylan Ex. 1031, pg 919