`Vol. 80, pp. 825-829, February 1983
`Immunology
`
`Immunoglobulin gene expression in transformed lymphoid cells
`
`Departments of *Genetics and tBiochemistry, Stanford University School of Medicine, Stanford. California 94305; and tDe
`Center. Institute of Cancer Reseuch, College of Physicians and Surgeons of Columbia University, New York, New York
`Contributed by Leonard A . Henenberg, October 15,1982
`
`ent of Microbiology and the Cancer
`
`ABSTRACX Myeloma, hybridoma, and thymoma cell lines
`have been successfully transfected for the Escherichia cdi xan-
`thine-guanine phosphoribosyltransferase gene (gpt) by using the
`plasmid vector pSV2-gpt. The transformed cells synthesize the
`bacterial enzyme $phospho-cr-wribose-1-diph0sphate:xanthine
`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
`transfeding cells with pSV2-gpt containing a rearranged K light
`chain genomic segment from the S107 myeloma cell line. The S107
`light chain is synthesized in gpt-transformed J558L myeloma cells
`and is identical to the light chain synthesized by the SlM myeloma
`cell line, as judged by immunoprecipitation and two-dimensional
`gel electrophoresis, Furthermore, this light chain is synthesized
`and secreted as part of an intact antibody molecule by transformed
`hybridoma cells that normally secrete an IgGl ( y , ~ ) 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
`genes have
`been conducted in heterologous host cells-i.e.,
`been transfected into cell types (particularly human HeLa and
`mouse L cells) that normally do not express the gene of interest
`(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 differentiation back into the cell type that nor-
`mally expresses the genes of interest. 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-hnction correlates of antibody molecules, we
`have explored techniques for transfeetion 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-a-D-ribose-.Jdiphosphate:xanthine phos-
`phoribosyltransferase, EC 2.4.2.22). Cells synthesizing XGPRT
`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 transformed cell line is hypoxanthine phos-
`phoribosyltransferase-negative (HPRT-; IMP pyrophosphate
`phosphoribosyltransferase, EC 2.4.2.8), transformants can be
`
`-
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`- -
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`The publication costs of this article were defrayed in part by page charge
`payment. This article must therefore be hereby marked 'hdwrtise-
`ment" in accordance with 18 U. S. C. 51734 solely to indicate this fact.
`
`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 fiom which the rearranged gene
`was isolated. Furthermore, in transformed hybridoma cells,
`this light chain is assembled with an immunoglobulin heavy
`chain and secreted as a complete antibody molecule.
`
`MATERIALS AND METHODS
`Cell Lines. J558L is a spontaneous heavy chain-loss-variant
`myeloma cell line obtained from the J558 cell line [a,A; anti-al-
`3 dextran (ll)] that synthesizes and secretes a A light chain. Y3-
`Ag1.2.3 is a HPRTrat myeloma cell line originally described
`by Galfre et a l (12) that synthesizes and secretes a rat K light
`chain. 27-44is a HPRT- mouse IgGl anti-dansyl hybridomacell
`line (13); and BW5147 is a HPRT-, ouabainr AKR thymoma
`originally described by Hyman and Stallings (14). Cell lines
`were maintained in either 10% newborn calf serum in Dul-
`becco's moddied minimal essential medium (DME medium) or
`10% fetal calf serum in alpha moddied 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 of this 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 J.-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
`cham 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-Goldin et d (9). &h-
`erichia coli K-12 strain HB101, containing the appropriate plas-
`mid, was grown at 3 P C in Luria broth containing 1% glucose
`to an absorbance at 600 nm of 0.6-0.8. Chloramphenicol was
`added to 125 pg/ml, the culture was incubated at 37OC for 12-
`16 hr to ampllfy 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 TrisHCl (pH 8) was added;
`
`Abbreviations: XGPRT. xanthine-manine phosphoribosyltransferase;
`
`HPRT, hypoxanthine phosphoribo~ltransf~rase;-~~~, h&xanthine/
`aminopterin/thymidine; DME medium, Dulbecco's modified minimal
`essential medium.
`
`Sanofi/Regeneron Ex. 1031, pg 915
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`
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`826
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`Immunology: Oi et oL
`
`Proc. Nod Acad. Sci. USA 80 (1983)
`
`Fro. 1. Structure of the vectora
`used for lymphoid cell transforma-
`tions. The dmgmm of the parental
`pSV2-gpt plasmid vector waa taken
`h m Mulligaa and Berg (4,s): pBR322
`DNA ie represented by the solid black
`linee and the plaamid'e DNA replica-
`tion origin and plactarnase gene are
`indicated, the gpt gene sequence is
`represented by the hatched segments;
`simian virua 40 (SV40) eequencea are
`the stippled segments. The SV40 ori-
`gin of DNA replication (on) and early
`promoter are loeated 5' of the gpt ee
`auencee. ~1sv2-mtTK~r has an ineer-
`6on of 24% ba&-&m; containing the
`herpea simplex thymidine kinase pro-
`moter, between the gpt gene and the
`SV40 early promoter (unpublished
`data). pSV2-S107 has a 7-kilobase
`BamHI fragment, containing the en-
`tire genomic S107 light chain gene,
`V,
`J~ inserted into the unique BamHI site
`of pSV2-gpt. This rearranged light
`chain gene is oriented in the oppoeite
`direction tagpt and cnntains the leader,
`V, and K constant redon exons as well
`flanking 5' and 3; sequences.
`
`until used, whereupon it was diluted to two times concentrated
`and adjusted to pH 7.05. Plasmid DNA (80 kg/ml) was made
`up in 125 mM CaClz which was stored as a 2 M stock solution
`at -20°C. DNA-calcium phosphate precipitates were formed
`by dropwise addition of the DNA into the HeBS solution. The
`precipitate formed in 30 rnin 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 ~hosphate precipitate
`(lo6 cells per 20 pg of DNA per 0.5 ml). This suspension was
`incubated at 37°C for 30 rnin and then was diluted 1:10 in
`serumcontaining medium. The cells were plated either into 24-
`well plates (2 x I d cells per well) or S w e l l plates (2 x lo4
`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 rnin at 3PC, serumcontaining 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 Gel Electrophoresis. Immuno-
`precipitations were done with [35S]methionine-labeled cell ly-
`sates and supernates. 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 NaDodSO,/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
`
`Em-apc
`
`the bacteria were suspended and 0.25 ml of lysozyme [a freshly
`prepared solution of 5 mg/ml in 0.25 M Tris-HC1 (pH 8)] was
`added. After 5 rnin of incubation 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-HC1
`(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 MgCl, that was warmed to 37% After further in-
`cubation for 10 rnin 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 O . S l x lo6 cells per
`ml in DME medium supplemented with 10% newborn calf
`serum. Five milliliters of the protoplast suspension was added
`to 2 x lo6 cells in growth medium. The mixture was centrifuged
`for 5 rnin at room temperature at approximately 500 X g. The
`supernate 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 CO,. After 3 rnin of centrifugation at 500 x g the poly-
`ethylene glycol was diluted with 7 ml of DME medium while
`resuspending the pellet. After 5 rnin of centrifugation at 500
`x g, the supernate 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 250
`pg/ml, hypoxanthine at 15 pg/ml, mycophenolic acid at 6 pg/
`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 transformants 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
`
`Sanofi/Regeneron Ex. 1031, pg 916
`
`
`
`Immunology: Oi et d
`
`Proc. Natl A d . Sci. USA 80 (1983)
`
`827
`
`Table 2. Transformation of lymphoid cell linee with the pSV2-
`gpt vectors by using calcium phosphate precipitation
`Cell line
`BW5147
`27-44
`Y3
`Vector
`1/192+
`10/192t
`3/48.
`pSV2-gpt
`1/192t
`10/192t
`19/48*
`pSVZ-gptTKpr
`O/192t
`43/288+
`47/48*
`pSV2S107
`Results are from three experiments with the Y3 cell line and two
`
`experiments with 27-44 and ~ ~ 5 1 4 7 celr lines.
`Cells were plated in Zbwell culture diehes at 2 x 10' cells per well.
`t Cells were platdin 96-well culture dishes at 4 x 10' cells per well.
`
`Ofthe four cell lines stably transformed with pSV2-S107, the
`J558L cell line synthesized, but did not secrete, the S107 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. Scharff, personal communication). Transformants of
`the 27-44 hybridoma cell line synthesized and secreted the S107
`light chain. Moreover, the S107 light chain was assembled into
`tetrameric H,L, immunoglobulin molecules with the endoge-
`nous yl heavy chain and 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 and gel analyses. XGPRT 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 J558L and 27-44 transfonnants are shown in Figs. 3
`and 4. The two-dimensional gel pattern of the S107 light chain
`synthesized by S107 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 transformants. 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 tnmsformants were im-
`munoprecipitated with both hybridoma anti-IgC1 allotypic an-
`tibody and rabbit anti-mouse light chain antisera. Both reagents
`precipitated the S107 light chain (data not shown). Sequenhal
`precipitation, first with the hybridoma anti-IgGl antibody and
`
`by every parameter tested. Different cell lines and different
`vectors produced different transhrmation frequencies. More-
`over, the two DNA delivery procedures, protoplast fusion and
`calcium phosphate precipitation, yielded different transfor-
`mation frequencies. Tables 1 and 2 summarize the results by
`using protoplast fusion and calcium phosphate precipitation,
`respectively.
`Under the present experimental conditions, BW5147 ap-
`pears to be the least competent recipient of the cell lines tested,
`Y3
`having a transformation frequency of approximately
`and 2744 yielded frequencies in the range of 0.3 to >5 x
`In the present experiments, J558L yielded the highest fre-
`quency with the range of 3 x
`to
`Protoplast fusion
`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 chaincontain-
`ing vector, pSV2-S107. This dramatic increase is evident when
`the pSV2-S107 vector was used with the J558L 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 2744 cell line was increased only
`about %fold with pSV2-S107. The sequence(s) in the pSVZ-Sl07
`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-gptTKpr than with pSV2-
`gpt (Table 2). Regardless of which vector was used, BW5147
`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 Werent
`(Fig. 2 and data not shown).
`XGPRT Activity. The transformed cell lines expressed the
`Eco gpt gene, as measured by the presence of XGPRT activity
`in the cell lysates. E. coli XGPRT can be distinguished from
`mammalian HPRT activity by its different electrophoretic mo-
`bility (4,5). In cellsselected for resistance to mycophenolic acid,
`both the cellular HPRT and bacterial XGPRT activities were
`detectable (Fig. 2). Cells lacking their own HPRT activity and
`selected for gpt in HAT medium had only the bacterial en y m e
`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 from 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 H z b
`tetramer. This also involves the formation of interchain disul-
`fide bonds.
`
`Table 1. Transformation of lymphoid cell lines with the
`pSV2-gpt vectors by wing protoplast fusion
`Cell line
`
`BW5147
`0/76* 1/48t
`0/80* 1/48t
`4/96. 8/48?
`
`Vector
`pSV2-gpt
`pSV2-gptTKpr
`pSV2S107
`
`J558L
`21/280* 27/36t
`10/190* 24/36t
`186/192* 36/36'
`149/192*
`Results are h r n three experiments and are expressed as the number
`of culture wells having stable tramformants.
`*After protoplast fuaion cells were plated in 96-well culture dishes at
`lo4 cells uer well.
`t Cells wek plated at 1@ cells per 2.0 ml of culture in 24-well dishes.
`*Cells were plated at 5 x loS cells per well in 96-well culture.dishes.
`
`FIG. 2. XGPRT and HPRT 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 tramformants; and 6 and 7, transformanta of
`the 27-44 cell line. Because 27-44 is a HPRT- cell line, only XGPRT
`is present.
`
`Sanofi/Regeneron Ex. 1031, pg 917
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`828
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`Immunology: Oi et d
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`Proc. Natl Acad. Sci. USA 80 (1983)
`
`FIG. 4. S107 light chain produced by transformed J558L cells. Au-
`toradiograms 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 twodimensional gels are shown. (A) A light
`chain produced by the parental J558L cell line. (B) S187 K light chain.
`(0 A mixture of the two light chains. The K and A light chains are
`distinguished on the basis of both charge and apparent Mr. W and E )
`Two independently derived J558L cell linee transformed with pSV2-
`5107 DNA. The transformant examined in D only appears to produce
`larger quantities of the 5107 K light chain than the endogenous 5558
`A light chain, because the S107 K chain is synthe8iza-I and remains in
`the cytoplasm, while the 5558 A chain is syntheeized and secreted.
`
`compared. Amounts varied from barely detectable to quantities
`equal to endogenous light chain. This variation may he due to
`the chromosomal region where the light chain has integrated.
`It also could result from different copy number of the light chain
`gene in dlfferent 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 determine the cause of this variation and why light
`chain expression does not occur in Y3 or BW5147 cell lines
`transformed 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 transformation 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
`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 S107 light chain is incorporated into the
`
`FIG. 3. 5107 light chain produd by transformed 27-44 cells. Au-
`toradiograms of two-dimensional gels of the light and heavy chains
`produced by parental and transformed cell lines are shown. (A) Pa-
`rental 27-44 IgGl anti-dansyl antibody immunoprecipitated with an
`anti-IgG1-specific hybridoma antibody. Both the yl heavy and K light
`chains can be seen. (B) 5107 IgA antibody immunoprecipitated with
`a rabbit anti-IgA antiserum. The a heavy chain was distinguished
`clearly by charge and apparent M, from the yl heavy chain in A. (0
`A mixture of the immunoprecipitates of A and B. The two K light
`chains can be seen as diatinct .spots (indicated by m w 8 ) having
`nearly identical charge but different apparent Mr. (D) Immunoprecip
`itate of a transformed 27-44 cell line. Only the yl 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 S107 light chain was secreted by these cells. This shows that
`the S107 light chain is assembled with the yl heavy chain into
`an intact antibody molecule.
`Different amounts of S107 light chains were produced when
`a number of independent J558L and 27-44 transformants were
`
`Sanofi/Regeneron Ex. 1031, pg 918
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`Immunology: Oi et d
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`Proc. NatL Acad. Sci. USA 80 (1983)
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`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 pglobin 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 hequency. Transcription hom the immunoglobulin
`promoter cannot be essential for the increased transfbrmation
`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 XGPRT 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 b r differential immunoglobulin
`gene expression at M e r e n t stages of lymphocyte differentia-
`tion. Cell lines in which immunoglobulin synthesis ca I 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 the cell contains the appropriate receptors
`(23).
`. .
`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 efficiently 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 immuaoglobuhn 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-
`graded in the Y3 myeloma has not been excluded.
`There is evidence that differentiated cell t ~ p e s exDress im-
`munoglobulin genes to varying levels. v or e x k p l e , somaticcell
`hybridization of myelomas yields hybridomas that produce an-
`tibodies, whereas thymomas yield hybrid cells wi&~-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 a t 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 het-erogeneous serum pools after immunization.
`The advent of myelomas, and more recently hybridomas, has
`permitted the s&dy of homogeneous populations of antibodies.
`DNA-mediated transfection and immunoglobulin gene expres-
`sion is an important tool to permit the study of immunogloiulin
`
`molecules. By using this technique, it should be possible to
`study the hnction of both novel chain combinations and novel
`chain structures. In oitro site-specific mutagenesis techniques
`can be used to construct specific mutations in immunoglobulin
`genes that can be expressed after transfecticm. Because signif-
`icant quantities of immunoglobulin are produced in the trans-
`formants, sufficient quantities of protein necessary for detailed
`analyses should be obtained.
`Note Added in Proof. After this paper was submitted for, publication,
`we learned 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 GM-132.35, 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