Proc. Natl. Acad. Sci. USA
`Vol. 80, pp. 6351-6355, October 1983
`Immunology
`
`Functional immunoglobulin M production after transfection of
`cloned immunoglobulin heavy and light chain genes into
`lymphoid cells
`(protoplast fusion/G418 selection)
`ATsuo Ocur*t, RoBERT G. HAWLEY*t, TERESA HAWLEY*t, MARC J. SuuLMANt+, ANDRE TRAUNECKER§,
`GEORGES KemLER§, AND NOBUMICHI HozuMr*t
`
`*Ontario Cancer Institute and tDepartment of Medical Biophysics, University of Toronto, 500 Sherbourne Street, Toronto, ON M4X 1K9 Canada;. *Rheumatic
`Disease Unit, Wellesley Hospital, Toronto, ON M4Y 1)3 Canada; and fBasel Institute for Immunology, Grenzacherstrasse 487, Basel CH-4005, SWitzerland
`
`Communicated by Niels Knj ]erne, july 11, 1983
`
`The rearranged immunoglobulin heavy (p.) and
`ABSTRACT
`light (K) chain genes cloned from the Sp6 hybridoma cell line pro(cid:173)
`ducing immunoglobulin M specific for the hapten 2,4,6-trinitro(cid:173)
`phenyl were inserted into the transfer vector pSV2-neo and in(cid:173)
`troduced into various plasmacytoma and hybridoma cell lines. The
`transfer of the p. and IC genes resulted in the production of pen(cid:173)
`tameric, hapten-specific, functional IgM.
`
`Work over the last decades has provided extensive information
`on immunoglobulin function and structure (1). Despite this in(cid:173)
`formation, it has been possible only in gross terms to relate mo(cid:173)
`lecular function with particular structural features.
`With the advent of genetic engineering and gene transfer
`techniques, questions regarding structure-function relation(cid:173)
`ships can now be readily addressed-that is, virtually any gene
`segment can be modified precisely in vitro and the novel seg(cid:173)
`ment can then be exchanged with its normal counterpart. By
`introducing such engineered genes into the appropriate cells,
`the effects of systematic alterations in protein structure on pro(cid:173)
`tein function can be assessed.
`Because immunoglobulin production is a specialized func(cid:173)
`tion of cells of the B-lymphocyte lineage, it is expected that the
`conditions for proper lg gene expression will be provided only
`in appropriate immunocompetent cells. For example, to pro(cid:173)
`duce normal pentameric lgM(K), a cell must transcribe, pro(cid:173)
`cess, and translate RNA for the 1.1. and K chains and also provide
`J protein, enzymes for the proper polymerization and glycosyla(cid:173)
`tion of the Ig chains, as well as a suitable secretory apparatus.
`We have previously described a system for transferring a func(cid:173)
`tional immunoglobulin K light chain gene into IgM-producing
`hybridoma cells (2). Here we extend this work to show that the
`transfer of the 1.1. and K chain genes of a defined specificity into
`various plasmacytoma and hybridoma cell lines results in the
`production of functional pentameric, hapten-specific IgM(K).
`
`MATERIALS AND METHODS
`Cell Lines. X63Ag8 was originally derived (3) from the plas(cid:173)
`macytoma MOPC21 and synthesizes lgG1(K) of unknown spec(cid:173)
`ificity. X63Ag8.653 was derived from X63Ag8 as a subclone that
`synthesizes neither the heavy (yl) nor light (K) chain (4). Sim(cid:173)
`ila1'ly, Sp2/0Ag14 is an lg nonproducing subclone of the Sp2
`hybridoma (5). Sp6 is a hybridoma making IgM(K) specific for
`the hapten 2,4,6-trinitrophenyl (TNP); originally this cell line
`produced the y1 and K chains of X63Ag8 as well as the (TNP
`specific) /.LTNP and KTNP chains (6). A subclone of Sp6 not mak-
`
`ing the y1 chain was isolated, and the Sp602 and Sp603 cell
`lines were derived from this y1 nonproducer. The mutant cell
`line igm-10, derived from Sp602 (7), lacks the gene encoding
`/.LTNP (8).
`Gene Transfer. The construction of pSV2-neo plasmid vec(cid:173)
`tors carrying the genes for /.LTNP or KTNP or both is described
`in the text. The vectors were transfected into the rk- mk- Esch(cid:173)
`erichia coli strain K803. To transfer the vector, bacteria bearing
`the appropriate plasmids were converted to protoplasts and fused
`to the indicated cell lines as described (2). The frequency of
`G418-resistant transformants per input cell was approximately
`10- 4 for X63Ag8 and Sp2/0Ag14, 10-5 for igm-10, and 10- 6 for
`X63Ag8.653.
`Analysis of lg. As described previously (7), lg was biosyn(cid:173)
`thetically labeled, in the presence or absence of tunicamycin,
`immunoprecipitated, and analyzed by NaDodS04/polyacryl(cid:173)
`amide gel electrophoresis with or without disulfide bond re(cid:173)
`duction. TNP binding IgM was assayed by TNP-dependent
`hemagglutination and by TNP-dependent enzyme-linked im(cid:173)
`munoadsorbent assay (ELISA) as described (2, 7). The hemo(cid:173)
`lyses of protein A-coupled erythrocytes and TNP-coupled
`erythrocytes were used to assay total lgM- and TNP-specific
`complement activating IgM, respectively (7).
`Analysis of RNA and DNA. Cytoplasmic RNA was isolated
`according to Schibler et al. (9) and subjected to RNA blot anal(cid:173)
`ysis as described by Thomas (10).
`Procedures for DNA extraction (ll), nitrocellulose blotting
`(12), and radiolabeling of probes (13) have been described (14,
`15). Probes specific for genes encoding immunoglobulin con(cid:173)
`stant and variable regions are detailed in the figure legends.
`
`RESULTS
`
`Description of Vectors and Expression Systems. The hy(cid:173)
`bridoma cell line Sp6 secretes IgM(K) specific for the hapten
`TNP. We have previously described the cloning of the TNP(cid:173)
`specific K gene, designated TK1 (16), and the construction of
`the recombinant, pR-TKl, where TK1 is inserted in the BamHI
`site of the vector pSV2-neo (2, 17). The /.LTNP gene was cloned
`in ACh4A from EcoRI partially digested DNA of Sp6 cells, and
`this clone is designated Sp6-718. The 16-kilobase-pair (kbp)
`fragment carrying the variable and constant regions was ob(cid:173)
`tained from Sp6-718 after partial digestion with EcoRI and was
`inserted at the EcoRI site of the vectors pSV2-neo and pR-TKl.
`In these recombinants, designated pR-Sp6 and pR-HLTNP• re-
`
`The publication costs of this article were defrayed in part by page charge
`payment. This article must therefore be hereby marked "advertise(cid:173)
`ment'' in accordance with 18 U.S.C. §1734 solely to indicate this fact.
`
`Abbreviations: TNP, 2,4,6-trinitrophenyl; ELISA, enzyme-linked im(cid:173)
`munoadsorbent assay; kbp, kilobase pair(s); SV40, simian virus 40; kb,
`kilobase(s).
`
`6351
`
`Sanofi/Regeneron Ex. 1040, pg 1021
`
`Merck Ex. 1040, pg 1047
`
`

`
`6352
`
`Immunology: Ochi et al.
`
`Proc. Natl. Acad. Sci. USA 80 (1983)
`
`spectively, the ILTNP gene lies in the same orientation as the
`KTNP gene in pR-TKl-i.e., the direction of transcription of ILTNP
`is opposite that of the simian virus 40 (SV40) early promoter
`(Fig. 1).
`The mutant cell lines igk-14 and igm-10 that lack the KTNP
`gene and ILTNP gene, respectively, were originally isolated from
`subclones of Sp6 (7). We have previously used igk-14 as are(cid:173)
`cipient cell line to assay expression of the KTNP gene (2).
`Expression of the ILTNP gene of pR-Sp6 was assayed here in
`igm-10. The simultaneous production of both /LTNP and KTNP
`chains from the vector pR-HLrnP is assayed in X63Ag8, the IgGl(cid:173)
`producing plasmacytoma parent of the Sp6 hybridoma. In later
`experiments the pR-HLn;p vector was assayed in the non(cid:173)
`producing cell lines Sp2/0Agl4 and X63Ag8.653. IgM pro(cid:173)
`duction by the transformants is compared with Sp603, a sub(cid:173)
`clone of the Sp6 hybridoma.
`Selection of IgM(K)-Positive Transformants. The recombi(cid:173)
`nant plasmid vectors bearing the lg genes also contain the bac(cid:173)
`terial gene neo, which renders the recipient cells resistant to
`
`VHtNP
`
`the antibiotic G418 (17). To transfer the Ig genes into the hy(cid:173)
`bridoma and plasmacytoma cells, bacteria harboring the re(cid:173)
`combinant plasmids were converted to protoplasts and fused
`with the various cell lines and G418-resistant cells were se(cid:173)
`lected. Depending on the cell line, the efficiency of G418-re(cid:173)
`sistant colonies ranged between 10-4 and 10-6 per input hy(cid:173)
`bridoma or plasmacytoma cell (see Materials and Methods). The
`culture supernatant of G418-resistant colonies was tested for
`TNP-specific IgM by using either a TNP-specific ELISA or by
`assaying agglutination ofTNP-coupled erythrocytes. In various
`experiments between 15% and 75% of the colonies were pos(cid:173)
`itive in such tests.
`Analysis of P.TNP and KTNP Production. Colonies that were
`positive for TNP-specific lgM were cloned by limiting dilution
`and examined further. The transformant IR44Ll, derived from
`the KTNp-positive cell line igm-10 and the ILTNP vector pR-Sp6,
`makes about 25% of the normal (Sp603) amount of lgM, as
`measured by the TNP-dependent ELISA. The transformant
`XR19U, derived from the cell line X63Ag8 and the ILTNP +
`KTNP vector pR-HLn;p, makes about 10% of the normal amount
`of IgM,
`To examine the /LTNP and KTNP separately, these chains were
`radiolabeled and analyzed by NaDodS04/polyacrylamide gel
`electrophoresis (Fig. 2). The Sp603 hybridomacell line still makes
`the K chain of its plasmacytoma parent, X63Ag8 (Fig. 2, lane
`a), as well as the specific ILTNP and KTNP chains (Fig. 2, lane
`e). The XR19U transformant derived from X63Ag8 has two ad(cid:173)
`ditional bands (Fig. 2, lane b), which comigrate with the ILTNP
`and KTNP of Sp603. The igm-10 cells used here make KTNP but
`have ceased to produce the K of X63Ag8 (Fig. 2, lane c), pre(cid:173)
`sumably because of a rearrangement in this K gene (see legend
`to Fig. 5). The IR44Ll transformant derived from igm-10 has
`one new band that comigrates with ILTNP (Fig. 2, lane d). As
`shown in Fig. 3, analysis of unreduced IgM by NaDodS04/
`polyacrylamide gel electrophoresis indicates that the trans(cid:173)
`fOrmants make predominantly pentameric IgM [(JL2K2)s].
`RNA Production. To examine the RNAs expressed by the
`transferred /LTNP and KTNP genes, cytoplasmic RNA from the
`transformants was fractionated by gel electrophoresis and probed
`
`d
`
`e
`
`b
`
`c
`
`..
`
`a
`
`fl.-
`
`y-
`
`EcoRI
`
`FIG. 1. Structure of the pR-Sp6 and pR-lll.nw plaamids. pR-Sp6
`contains the functionally rearranged /LTNP gene (""16 kbp), which was
`inserted into the EcoRI site of pSV2-neo (see text). In addition to the
`/LTNP gene, pRlii.nw contains the functionally rearranged KTNP gene
`(9.6 kbp) at the .Bam.HI site (2). lg genes are represented by heavy dark
`lines. The directions of transcription of the lg genes and the SV 40 early
`region are indicated by arrows. The p. and K exons are shown as filled
`boxes. M denotes alternative COOH-terminal coding regions that are
`utilized in the synthesis of membrane IgM. Thin lines are of pBR322
`origin. The white boxes denote DNA derived from SV40, into which the
`bacterial gene conferring neomycin resistance (hatched box) has been
`inserted. For specific details concerning the pSV2-neo transfer vector
`(donated by P. Berg), see ref. 17.
`
`FIG. 2. Analysis of heavy and light chains of secreted lg. G418-
`resistant transformant clones were biosynthetically radiolabeled with
`1.C]leucine as described (7). Secreted immunoglobulins were immu(cid:173)
`[
`noprecipitated with rabbit anti-mouse lgM antibody complexed with
`protein A-Sepharose CL-4B beads (Pharmacia). The precipitated ma(cid:173)
`terial was reduced with 2-merca.ptoethanol and analyzed by electro(cid:173)
`phoresis on a NaDodSO./polyacrylamide gel. Lane a, X63Ag8; lane b,
`XR19IA; lane c, igm-10; lane d, IR44L1; and lane e, wild-type hybri(cid:173)
`domaSp603.
`
`Sanofi/Regeneron Ex. 1040, pg 1022
`
`Merck Ex. 1040, pg 1048
`
`

`
`Immunology: Ochi et al.
`
`Proc. Natl. Acad. Sci. USA 80 (1983)
`
`6353
`
`c
`
`d
`
`e
`
`a
`
`b
`
`• iii II
`--
`
`FIG. 3. Analysis of secreted (unreduced) lg. The radiolabeled cul(cid:173)
`ture supernatants as described in the legend to Fig. 2 were analyzed by
`electrophoresis on a NaDod.S04/polyacrylamide gel without reducing
`the disulfide bonds (7). Lane a, X63Ag8; lane b, XR191A; lane c, igm-
`10; lane d, ffi44Ll; and lane e, wild-type hybridoma Sp603. The mark(cid:173)
`ers indicate the major forms of Sp603 IgM and X63Ag8 IgGl.
`
`with various ,_,. and K-specific DNA sequences (Fig. 4). RNA
`for the 1-t heavy chain was detected with a probe from the C,.4
`region. The transformants XR19IA and IR44Ll have bands at
`both 2. 7 and 2.4 kilo bases (kb), whereas the parental hybridoma
`Sp603 has only one band at 2.4 kb (Fig. 4A). A genomic probe
`containing the ,_, membrane-specific exon hybridized only to
`the 2. 7-kb band (data not shown). RNAs of 2. 7 and 2.4 kb have
`been found to encode the membrane !.J.tm) and secreted (p,.) forms
`of the 1-t chain, respectively (19-21). These results suggest that,
`whereas Sp603 makes RNA only for the p,. fonn, the trans(cid:173)
`formants make RNAs for both 14n and p,.. However, we have
`been unable to detect membrane lgM by staining with flu(cid:173)
`orescent I-t-specific antibodies. The 14n form has a longer poly(cid:173)
`peptide chain than does the p,. form and consequently can be
`distinguished from p,. by its lower mobility in NaDodS04/
`polyacrylamide gel electrophoresis. Therefore, we examined
`intracellular 1-t chains that were biosynthetically radio labeled in
`the presence of tunicamycin; for each transfonnant we found
`only one 1-t band, and this band comigrated with the 1-t band of
`Sp6 (results not shown). These observations suggest that either
`the 2. 7-kb RNA is not translated or that the 14n protein is very
`short-lived in the transfonnants.
`In a similar manner, the RNA blots were hybridized with a
`probe derived from the KTNP V region. Compared to Sp603 and
`igm-10, the transfonnant XR19IA was found to make a low
`amount of a 1.2-kb RNA that comigrated with authentic KTNP
`RNA (Fig. 48).
`Structure of Transferred DNA. To analyze the organization
`of the transferred pR-Sp6 and pR-HLTNP plasmids in the trans(cid:173)
`formed cell lines, BamHI-digested cell DNA was hybridized
`with probes specific for the ~-t· and K-chain constant region gene
`segments. The C,.1-2 probe used here spans the BamHI re(cid:173)
`striction site in the C,.2 exon (Fig. 1). Therefore, a minimum
`of two fragments is expected to be detected with this probe.
`
`A
`
`kt
`
`a
`
`b
`
`c
`
`d
`
`e
`
`2.7-
`24-t
`
`•
`
`..
`
`8
`
`a
`
`b
`
`c
`
`d e
`
`kb
`
`1.2- _ . . - -
`
`FIG. 4. Detection of iJ.TNP and KTNP gene sequences in cytoplasmic
`RNA from transformed cell lines. Lanes a, X63Ag8; lanes b, XR19L4;
`lanes c, igm-10; lanes d, ffi44Ll; and lanes e, Sp603. Ten micrograms
`of total cytoplasmic RNA (9) was denatured with glyoxal, electroph(}(cid:173)
`resed through a horizon tall% agarose gel in 10 mM sodium phosphate
`buffer at pH 6.9, and transferred to nitrocellulose as described by Thomas
`(10). (A) The blot was hybridized with a 32P-labeled probe correspond(cid:173)
`ing to the C~4 exon. This probe was isolated from the eDNA clone
`pH76p.l7 (donated by J. Adams) after digestion with Pst I (18). (B) A
`similar blot was hybridized with a 32P-labeled probe containing KTNP
`V-region coding sequences (16). Sizes were estimated by comparison to
`mouse ribosomal28S and 188 RNA (4.7 and 2.0 kb, respectively).
`
`Two fragments of 6.0 and 16 kbp were detected in the DNA
`of both of the transfonnants. These correspond to the frag(cid:173)
`ments generated by BamHI digestion of the intact pR-Sp6 and
`pR-HLTNP plasmids (Fig. 5). In addition, one (XR19IA) or two
`(IR44Ll) extra fragments could be detected in the DNA from
`these cell lines. In parallel experiments, sequences indicative
`of unintegrated pR-TKl plasmids have not been detected in the
`low molecular weight fraction of the Hirt supernatants (25) of
`similarly transformed igk-14 cells (results not shown). Taken
`together, these results suggest that the transferred genes are
`tandemly integrated into the chromosomal DNA of the recip(cid:173)
`ient cells.
`
`Sanofi/Regeneron Ex. 1040, pg 1023
`
`Merck Ex. 1040, pg 1049
`
`

`
`6354
`
`Immunology: Ochi et al.
`
`Proc. Natl. Acad. Sci. USA·80·(1983)
`
`Table 1. Assay of functionallgM
`
`Phenotype
`lgM, K(TNPl
`+ K(X63)
`K(TNP)
`
`IgG1, K
`
`Nolg
`
`Cell line
`Sp603
`
`igm-10
`ffi44L1
`
`X63Ag8
`XR19L4
`
`Sp2j0Ag14
`SR1.2
`SR40.1
`
`X63Ag8.653 Nolg
`X653Rl.l
`
`Hemolysis titer
`on erythrocytes
`coupled with
`Protein A
`2"
`
`TNP
`26
`
`TNP /protein
`A ratio
`4
`
`<1
`23
`
`<1
`23
`
`<1
`24
`2
`
`<1
`24
`
`<1
`2~
`
`<1
`<1
`
`<1
`2"
`22
`
`<1
`26
`
`4
`
`<1:8
`
`4
`2
`
`4
`
`As described in the text, the transformants ffi44Ll and XR19L4 were -
`derived by introducing the IJ.TNP gene alone or the ILTNP and KTNP genes
`together into the igm-10 and X63Ag8 cell lines. Similarly, the cell lines
`SR1.2, SR40.1, and X653R1.1 were generated by transferring the /LTNP
`+ KTNP vector pR-~ into Sp2f0Ag14 and X63Ag8.653. The indi(cid:173)
`cated cell lines were grown to approximately 106 cells per ml; and cul(cid:173)
`ture supernatants were assayed for lgM .concentration (lysis titer on
`protein A-coupled erythrocytes) and TNP-specific hemolysis activity
`(lysis titer on TNP-coupled erythrocytes). Culture supernatants were ·
`diluted serially 1:2 to obtain the end-point dilution (titer) that still caused
`lysis. The ratio of the TNP and the protein A titer is a measure of the
`specific activity of the secreted lgM.
`
`to other fragments that correspond to the K chain genes en(cid:173)
`dogenous to the recipient X63Ag8·cellline (23, 24).
`Assay of IgM. Function. We have tested the normal ftmc(cid:173)
`tioning of the lgM produced by the transformants by assaying
`its action in complement..Jependent lysis of TNP-coupled
`erythrocytes (Table 1). The lgM concentration in the culture
`supernatants of. the indicated cell lines was measured. by the
`hemolysis of protein A-coupled erythrocytes in the presence of
`anti-1gM (7). These results indicate that lgM made by 1R44Ll
`has normal activity with regard to TNP binding and comple(cid:173)
`ment activation. However, the transfurmant XR19lA makes lgM
`that has an activity that is less than 1/30th of the normal activity
`in the TNP-dependent hemolysis assay. X63Ag8 still produces
`the myeloma K chain, and this K chain can be incorporated into
`lgM, thus reducing TNP-specific hemolysis activity (7). To avoid
`this problem of the nonspecific myeloma K chain, the ILTNP +
`KTNP vector pR-HLTNP was transferred into the·nonproducer
`cell lines Sp2/0Ag14 (5) and X63Ag8.653 (4). The. lgM pro(cid:173)
`duced by transformants of these cell lines has normal activity
`for TNP-specific hemolysis (Table 1).
`
`DISCUSSION
`
`We and others have previously reported the expression of lg
`light chain genes in various cell types (2, 26-29). In this paper
`we have described the construction of plasmids that bear genes
`for TNP-specific immunoglobulin IL and K chains. The expres(cid:173)
`sion of these genes was studied after the transferof the plas(cid:173)
`mids into various cell lines derived from lg-secreting plasma(cid:173)
`cytomas or hybridomas. The transfer of these plasmids into these
`cells is usually (see below) sufficient to cause the production of
`pentameric lgM(K) that binds antigen (TNP) and activates com(cid:173)
`plement"'-that is, these cell lines (X63Ag8, X63Ag8.653, igm-
`10, and Sp2/0Ag14) provide all of the machinery necessary for
`lgM production except the structural genes for the IL and K
`
`A
`
`0
`
`b
`
`c d
`
`e
`
`f
`
`- -
`
`~.bp
`
`15
`
`6.0
`
`8
`
`a
`
`b
`
`c
`
`d
`
`e
`
`f
`
`kbp
`
`96-
`
`69
`59-;
`54
`
`----·-
`
`FIG. 5. Detection of pR-Sp6 and pR-HLTNP sequences in DNA from
`transformed cell lines. Lanes a, X63Ag8; lanes b, XR19L4; lanes c, igm-
`10; lanes d, ffi44L1, lanes.e, Sp603; and lanes f, igm-10 with 5 equiv(cid:173)
`alents of pR-Sp6. Bamill-digested DNA samples (20 p.g) were electro(cid:173)
`phoresed through a 1% agarose gel at 2 V "CIIl- 1 for 40 hr and transferred
`to nitrocellulose. (A) A previously hybridized blot (see B) was washed
`according to Thomas (10) and rehybridized to a 32P-labeled probe con(cid:173)
`taining the C~1 and C~2 exons. This probe was prepared by isolation of
`an appropriate fragment from a Xba IjHindill digestion of a genomic
`clone of the IL-chain constant region gene segment. The bands corre(cid:173)
`sponding to the ,.-chain gene-containing fragments generated by Bamill
`digestion of pR-Sp6 and pR-HLTNP are indicated. The two bands ob(cid:173)
`served in lane e (11 and 14 kbp) correspond to the functionally rear(cid:173)
`ranged ILTNP gene in the wild-type Sp603 cell line. (B) The same blot
`was hybridized with a 32p-labeled probe containing the K-constant re(cid:173)
`gion gene .segment that was isolated from the plasmid pL21-5 (donated
`by R. Wall) (22). The bands at 9.6 kb correspond to the "TNP gene (16).
`The bands at 6.9, 5.9, and 5.4 kbp correspond to rearranged K chain genes
`present in the DNA of the X63Ag8 cell line (23, 24), two of which (5.9
`and 5.4 kbp) were retained in the generation of the original Sp6 hy(cid:173)
`bridoma. The 5.4-kbp band corresponds to the functionally rearranged
`X63Ag8 K gene and this band is not observed in the case of igm-10 (lane
`c). Sizes were estinlated by comparison toHindlll-digested A phage DNA.
`
`The pattern obtained for XR19lA upon hybridization of the
`same blot with the c. probe is consistent with the above inter(cid:173)
`pretation. DNA from this transformant contains a 9.6-kbp frag(cid:173)
`ment corresponding to the wild-type KTNP gene (16) in addition
`
`Sanofi/Regeneron Ex. 1040, pg 1024
`
`Merck Ex. 1040, pg 1050
`
`

`
`Immunology: Ochi et al.
`
`Proc. Natl. Acad. Sci. USA 80 (1983)
`
`6355
`
`chains. The capacity to provide this machinery is present de(cid:173)
`spite the fuct that these cell lines have been propagated for years
`without overt selection for this property.
`We expect that this system will be very useful in determining
`the structural requirements for normal lgM production and
`function. To date, the use of genetics for this purpose has been
`limited to the analysis of naturally occurring mutants that in(cid:173)
`terfere with normal IgM processing and activity (7, 30). Al(cid:173)
`though such mutanls are useful as a starting point, in vitro mu(cid:173)
`tagenesis offers a more rapid and systematic method of obtaining
`altered IgM. Thus, it should be possible to identify the amino
`acids that are critical for complement activation or Fe receptor
`binding. Similarly, one can expect to define the features that
`are necessary for pen tamer formation, glycosylation, and se(cid:173)
`cretion.
`As is the case with other gene transfer systems, we have found
`that the various transformants produce quite different amounts
`of !L and K chain, ranging from undetectable to approximately
`normal levels. In general, a linear relationship doos not exist
`between the copy num her of the transferred sequences and the
`level of lg gene expression. Studies with transfer vectors pre(cid:173)
`sumed to be replication incompetent indicate that the trans(cid:173)
`ferred sequences integrate into different sites in the host chro(cid:173)
`mosomes, independent of the method of transfer (31-33).
`Therefore, the context of the transferred genes is different from
`normal and different in each Iecipient. It is not known whether
`it is the different chromosomal locales that are responsible for
`the variation in the expression of the transferred genes or whether
`these results reflect a high frequency of mutation associated
`with the introduction of exogenous DNA into mammalian cells
`(34, 35).
`The transformants XR19L4 and IR44L1 produce, in addition
`to a 2.4-kb RNA that comigrates with authentic ILs RNA, a 2. 7-
`kb RNA that appears to include the f.Lm exon. As we have been
`unable to detect a f.Lm protein, it is possible that the 2. 7 -kb RNA
`is aberrant in some respect (36-39). In contrast to the heavy
`chain gene results, the transferred K chain genes in XR19L4
`and in several transformants derived from igk-14 and the KTNP
`vector pR-TKl (ref. 2; unpublished data) produce a single spe(cid:173)
`cies of RNA that comigrates with authentic KTNP RNA.
`We expect that the variations in the expression of the trans(cid:173)
`ferred genes will not interfere with the usefulness of this sys(cid:173)
`tem in producing altered lgM for functional analysis. Further(cid:173)
`more, we anticipate that modifications of this protocol will allow
`investigation of the mechanisms controlling Ig gene expression.
`
`Note Added in Proof. Gillies et al. (40) and Neuberger (41) have re(cid:173)
`cently reported the expression of cloned heavy chain genes in trans(cid:173)
`formed lymphoid cells.
`
`We thank Nusrat Govindji and Catherine Filkin for expert technical
`assistance. This work was supported by grants from the Medical Re(cid:173)
`search Council, the National Cancer Institute, the Arthritis Society, the
`Allstate Foundation, and Hoffmann-La Roche Ltd. A.O. was sup(cid:173)
`ported by a Terry Fox Cancer Research Fellowship from the National
`Cancer Institute. R.G.H. was supported by a studentship from the
`Medical Research Council.
`
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`Sanofi/Regeneron Ex. 1040, pg 1025
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`Merck Ex. 1040, pg 1051