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/0! 18 selection)
`ATSUO OCHr*T, ROBERT C. HAwLEY*T, TERESA HAwLEY*T, MARC J. SHULMANl¢, ANDRE TRAUNECKER§,
`GEORGES KorrLER§, AND NOBUMICHI HOZUMI*l
`*Ontario Cancer Institute and lDepartm
`ent of Medical Biophysics, University of Toronto, 500 Sherboume Street, Toronto, ON M4X 1K9 Canada; ‘Rheumatic
`; and §Basel Institute for Immunology, Crenzacherstrasse 487, Basel CH-4005, Switzerland
`Disease Unit, Wellesley Hospital, Toronto, ON M4Y 1J3 Canada
`
`Communicated by Niels Kaj Jerne, July 11, 1983
`
`The rearranged immunoglobulin heavy (u) and
`ABSTRACT
`light (K) chain genes cloned from the Sp6 hybridoma cell line pro-
`ducing immunoglobulin M specific for the hapten 2,4,6-trinitro-
`phenyl were inserted into the transfer vector pSV2-neo and in-
`troduoed into various plasmacytoma and hybridoma cell lines. The
`transfer of the u and K genes resulted in the production of pen-
`tameric, hapten-specific, functional IgM.
`
`Work over the last decades has provided extensive information
`on immunoglobulin function and structure (1). Despite this in-
`formation, it has been possible only in gross terms to relate mo-
`lecular function with particular structural features.
`With the advent of genetic engineering and gene transfer
`techniques, questions regarding structure—function relation-
`ships can now be readily addressed—that is, virtually any gene
`segment can be modified precisely in vitro and the novel seg-
`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-
`tein function can be assessed.
`Because immunoglobulin production is a specialized func-
`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-
`duce normal pentameric IgM(K), a cell must transcribe, pro-
`cess, and translate RNA for the y. and K chains and also provide
`J protein, enzymes for the proper polymerization and glycosyla-
`tion of the Ig chains, as well as a suitable secretory apparatus.
`We have previously described a system for transferring a func-
`tional immunoglobulin K light chain gene into IgM-producing
`hybridoma cells (2). Here we extend this work to show that the
`transfer of the ,u 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-
`macytoma MOPC21 and synthesizes lgGl(K) of unknown spec-
`ificity. X63Ag8.653 was derived from X63Ag8 as a subclone that
`synthesizes neither the heavy (yl) nor light (K) chain (4). Sim-
`ilarly, Sp2/0Ag14 is an lg nonproducing subclone of the Sp2
`hyhridoma (5). Sp6 is a hybridoma making IgM(K) specific for
`the hapten 2,4,6-trinitrophenyl (TNP); originally this cell line
`produced the yl and K chains of X63Ag8 as well as the (TNP
`specific) p.-mp and KTNp chains (6). A subclone of Sp6 not mak-
`
`The publication costs of this article were defrayed in part by page charge
`payment. This article must therefore be hereby marked “advertise-
`rnent" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
`
`6351
`
`ing the yl chain was isolated, and the Sp602 and Sp603 cell
`lines were derived from this 71 nonproducer. The mutant cell
`line igm-10, derived from Sp602 (7), lacks the gene encoding
`P«'rNr (3)-
`Gene Transfer. The construction of pSV2-neo plasmid vec-
`tors carrying the genes for p.-mp or K1'N}) or both is described
`in the text. The vectors were transfected into the rfmk" Esch-
`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 Sp2/0Agl4, 10-5 for igm-10, and 10-6 for
`X63Ag8.653.
`Analysis of Ig. As described previously (7), lg was biosyn-
`thetically labeled, in the presence or absence of tunicamycin,
`immunoprecipitated, and analyzed by NaDodSO4/polyacryl-
`amide gel electrophoresis with or without disulfide bond re-
`duction. TNP binding IgM was assayed by TNP-dependent
`hemagglutination and by TNP-dependent enzyme-linked im-
`munoadsorbent assay (ELISA) as described (2, 7). The hemo-
`lyses of protein A-coupled erythrocytes and TNP-coupled
`erythrocytes were used to assay total IgM- and TNP-specific
`complement activating IgM, respectively (7).
`Analysis of RNA and DNA. Cytoplasmic RNA was isolated
`according to Schihler et al. (9) and subjected to RNA blot anal-
`ysis as described by Thomas (10).
`Procedures for DNA extraction (11), nitrocellulose blotting
`(12), and radiolabeling of probes (13) have been described (14,
`15). Probes specific for genes encoding immunoglobulin con-
`stant and variable regions are detailed in the figure legends.
`
`RESULTS
`
`Description of Vectors and Expression Systems. The hy-
`bridoma cell line Sp6 secretes IgM(K) specific for the hapten
`TNP. We have previously described the cloning of the TNP-
`specific K gene, designated TK1 (16), and the construction of
`the recombinant, pR-TK1, where TK1 is inserted in the BamHI
`site of the vector pSV2-neo (2, 17). The IJTNP gene was cloned
`in ACh4A from EcoRl 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-
`tained from Sp6—718 after partial digestion with EcoRl and was
`inserted at the EcoRI site of the vectors pSV2-neo and pR-TK1.
`In these recombinants, designated pR-Sp6 and pR-HL-mp, re-
`
`Abbreviations: TNP, 2,4,6-trinitrophenyl; ELISA, enzyme-linked im-
`munoadsorbent assay; kbp, kilobase pair(s); SV40, simian virus 40; kb,
`kilobase(s).
`
`Sanofi/Regeneron Ex. 1040, pg 1021
`
`Mylan Ex. 1040, pg 1021
`
`

`
`6352
`
`Immunology: Ochi et al.
`
`Proc. Natl. Acad. Sci. USA 80 (1983)
`
`spectively, the amp gene lies in the same orientation as the
`KTN]: gene in pR-TK1—i.e., the direction of transcription of lamp
`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 K-mp
`gene and amp gene, respectively, were originally isolated from
`subclones of Sp6 (7). We have previously used igk-14 as a re-
`cipient cell
`line to assay expression of the K'fNp gene (2).
`Expression of the ].L1'Np gene of pR-Sp6 was assayed here in
`igm-10. The simultaneous production of both ump and K-mp
`chains from the vector pR-HL-mp is assayed in X63Ag8, the IgCl-
`producing plasmacytoma parent of the Sp6 hybridoma. In later
`experiments the pR-HL-mp vector was assayed in the non-
`producing cell lines Sp2/0Agl4 and X63Ag8.653. IgM pro-
`duction by the transformants is compared with Sp603, a sub-
`clone of the Sp6 hybridoma.
`Selection of IgM(K)-Positive Transformants. The recombi-
`nant plasmid vectors bearing the Ig genes also contain the bac-
`terial gene neo, which renders the recipient cells resistant to
`
`EcoRI
`
`VHTNP
`
`
`EcoRI
`
`the antibiotic C418 (17). To transfer the Ig genes into the hy-
`hridoma and plasmacytoma cells, bacteria harboring the re-
`combinant plasmids were converted to protoplasts and fused
`with the various cell lines and G418-resistant cells were se-
`lected. Depending on the cell line, the elficiency of G418-re-
`sistant colonies ranged between 10"‘ and 10-6 per input hy-
`bridoma or plasmacytoma cell (see Materials and Methods). The
`culture supernatant of C418-resistant colonies was tested for
`TNP-specific IgM by using either a TNP-specific ELISA or by
`assaying agglutination of TNP-coupled erythrocytes. In various
`experiments between 15% and 75% of the colonies were pos-
`itive in such tests.
`Analysis of pm,» and Km; Production. Colonies that were
`positive for TNP-specific IgM were cloned by limiting dilution
`and examined further. The transformant IR44Ll, derived from
`the K1-Np-positive cell line igm-10 and the ,u.-mp vector pR-Sp6,
`makes about 25% of the normal (Sp603) amount of IgM, as
`measured by the TNP-dependent ELISA. The transformant
`XRl9L4, derived from the cell line X63Ag8 and the ;1.1-Np +
`K-[Np vector pR-Hlrmp, makes about 10% of the normal amount
`of IgM,
`To examine the amp and K'fNp separately, these chains were
`radiolabeled and analyzed by NaDodSO4/polyacrylamide gel
`electrophoresis (Fig. 2). The Sp603 hybridoma cell line still makes
`the K chain of its plasmacytoma parent, X63Ag8 (Fig. 2, lane
`a), as well as the specific p.-mp and K1-Np chains (Fig. 2, lane
`e). The XR19L4 transformant derived from X63Ag8 has two ad-
`ditional bands (Fig. 2, lane b), which comigrate with the 1.1.-mp
`and K-mp of Sp603. The igm-10 cells used here make K-[Np but
`have ceased to produce the K of X63Ag8 (Fig. 2, lane c), pre-
`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 pump (Fig. 2, lane d). As
`shown in Fig. 3, analysis of unreduced IgM by NaDodSO4/
`polyacrylarnide gel electrophoresis indicates that the trans-
`formants make predominantly pentameric IgM [([.l.2K2)5].
`RNA Production. To examine the RNAs expressed by the
`transferred amp and K'1'Np genes, cytoplasmic RNA from the
`transformants was fractionated by gel electrophoresis and probed
`
`0
`
`b
`
`c
`
`d
`
`e
`
`#- Q T
`
`7-«X-mm
`
`FIG. 1. Structure of the pR-Sp6 and pR-HL-mp plasmids. pR-Sp6
`contains the functionally rearranged [J/[Np gene (~16 kbp), which was
`inserted into the Eco}?! site of pSV2-neo (see text). In addition to the
`p.-mp gene, pRl{L-mp contains the functionally rearranged Kfiflp gene
`(9.6 kbp) at the Bamlll site (2). lg genes are represented by heavy dark
`lines. The directions oftranscription of the Ig genes and the SV40 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). Bee ref. 17.
`
`K‘ Ham» 3
`TNP
`
`FIG. 2. Analysis of heavy and light chains of secreted lg. G418-
`resistant transformant clones were biosynthetically radiolabeled with
`[“C]leucine as described (7). Secreted immunoglobulins were immu-
`noprecipitated with rabbit anti-mouse IgM antibody complexed with
`protein A-Sepharose CL-4B beads (Pharmacia). The precipitated ma-
`terial was reduced with 2-mercaptoethanol and analyzed by electro-
`phoresis on a NaDodSO4/polyacrylamide gel. Lane a, X63Ag8; lane b,
`XR19L4; lane c, igm-10; lane d, IR44L1; and lane e, wild-type hybri-
`doma Sp603.
`
`Sanofi/Regeneron Ex. 1040, pg 1022
`
`Mylan Ex. 1040, pg 1022
`
`

`
`Immunology: Ochi et al.
`
`Proc. Natl. Acad. Sci. USA 80 (1983)
`
`6353
`
`abcde
`
`A
`
`abode
`
`kl:
`
`{;,t2;<rNP2)5 {
`
`ptzxmez _,
`
`1 ,_,,,.,
`
`ya/<2-> 1 --
`).u<mP ->
`
`FIG. 3. Analysis of secreted (unreduced) Ig. The radiolabeled cul-
`ture supernatants as described in the legend to Fig. 2 were analyzed by
`electrophoresis on a NaDodSO4/polyacrylamide gel without reducing
`the disulfide bonds (7). Lane a, X63Ag8; lane b, XR19L4; lane c, igm-
`l0; lane d, IR44L1; and lane e, wild-type hybridoma Sp603. The mark-
`ers indicate the major forms of Sp603 IgM and X63Ag8 IgG1.
`
`with various ;1.- and K-specific DNA sequences (Fig. 4). RNA
`for the )1. heavy chain was detected with a probe from the C“4
`region. The transformants XR19L4 and IR44Ll have bands at
`both 2. 7 and 2.4 kilobases (kb), whereas the parental hybridoma
`Sp603 has only one band at 2.4 kb (Fig. 4A). A genomic probe
`containing the p. 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 ([4,...) and secreted (/1.5) forms
`of the [L chain, respectively (19-21). These results suggest that,
`whereas Sp603 makes RNA only for the us form, the trans-.
`formants make RNAS for both 14.," and us. However, we have
`been unable to detect membrane IgM— by staining with flu-
`orescent ;.L-specific antibodies. The ,u.m form has a longer poly-
`peptide chain than does the [L5 form and consequently can be
`distinguished from us by its lower mobility in NaDodSO4/
`polyacrylamide gel electrophoresis. Therefore, we examined
`intracellular p. chains that were biosynthetically radiolabeled in
`the presence of tunicamycin; for each transforrnant we found
`only one )1. band, and this band comigrated with the pl. band of
`Sp6 (results not shown). These observations suggest that either
`the 2.7-kb RNA is not translated or that the it". protein is very
`short-lived in the transformants.
`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 transformant XRIQLA was found to make a low
`amount of a 1.2-kb RNA that comigrated with authentic K1-Np
`RNA (Fig. 43).
`Structure of Transferred DNA. To analyze the organization
`of the transferred pR-Sp6 and pR-HL1-Np plasmids in the trans-
`formed cell lines, Baml-II-digested cell DNA was hybridized
`with probes specific for the p.- and K-chain constant region gene
`segments. The C,.1—2 probe used here spans the BamHI re-
`striction site in the C,‘2 exon (Fig. 1). Therefore, a minimum
`of two fragments is expected to be detected with this probe.
`
`kb
`
`l-2_
`
`....mB
`
`FIG. 4. Detection of [L1'Np and KTNp gene sequences in cytoplasmic
`RNA from transformed cell lines. Lanes a, X63Ag8; lanes b, XR19L4;
`lanes c, igm-10; lanes d, IR44L1; and lanes e, Sp603. Ten micrograms
`of total cytoplasmic RNA (9) was denatured with glyoxal, electropho-
`resed through a horizontal 1% 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 "P-labeled probe correspond-
`ing to the C,,4 exon. This probe was isolated from the cDNA clone
`pH76p.17 (donated by J. Adams) after digestion with Pst I (18). (B) A
`similar blot was hybridized with a “P-labeled probe containing K-mp
`V-region coding sequences (16). Sizes were estimated by comparison to
`mouse ribosomal 28S and 18S 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 transformants. These correspond to the frag-
`ments generated by BamHI digestion of the intact pR-Sp6 and
`pR-HL-mp plasmids (Fig. 5). In addition, one (XRl9L4) or two
`(IR44L1) extra fragments could be detected in the DNA from
`these cell lines. In parallel experiments, sequences indicative
`of unintegrated pR-TK1 plasmids have not been detected in the
`low molecular weight fraction of the Hirt supematants (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-
`ient cells.
`
`Sanofi/Regeneron Ex. 1040, pg 1023
`
`Mylan Ex. 1040, pg 1023
`
`

`
`6354
`
`Immunology: Ochi et al.
`
`Proc. Natl. Acad. Sci. USA 80 1983)
`
`A
`
`:3
`
`b
`
`c
`
`d
`
`e
`
`1‘
`
`K00
`
`Table 1. Assay of functional IgM-
`Hemolysis titer
`on erythrocytes
`
`-wmaael
`
`”'“" ~
`
`N“
`
`Islands
`
`6.0» W
`
`""""
`
`kbp
`
`
`
`FIG. 5. Detection of pR-Sp6 and pR-HL-mp sequences in DNA from
`transformed cell lines. Lanes a, X63Ag8; lanes b, XR19L4; lan c, igm-
`10; lanes d, lR44L1, lanes .e, Sp603; and lanes f, igm-10 with 5 equiv-
`alents of pR-Sp6. Bamfll-digested DNA samples (20 pg) were electro-
`phoresed through a 1% agarose gel at 2 V-cm‘ 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-
`taining the C,,1 and C,,2 exons. This probe was prepared by isolation of
`an appropriate fragment from a Xba I/HindlII digestion of a genomic
`clone of the p,-chain constant region gene segment. The bands corre-
`sponding to the iochain geneoontaining fragments generated by Baml-ll
`digestion of pR-Sp6 and PR-l'[L-1-Np are indicated. The two bands ob-
`served in lane e (11 and 14 kbp) correspond to the functionally rear-
`ranged [1-1'N'p gene in thewild-type Sp603 cell line. (B) The same blot
`was hybridized with a “P-labeled probe containing the K-constant re-
`gion genesegment that was isolated from the plasmid pL21-5 (donated
`by R. Wall) (22). The bands at 9.6 kb correspond to the K1-Np gene (16).
`The bandsat 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-
`bridoma. The 5.4-kbp band corresponds to the functionally rearranged
`X63Ag8 K gene and this band is not observed in the case of ig'm-10 (lane
`c). Sizes were estimated by comparison to HindI[I-digested A phage DNA.
`
`The pattern obtained for XR19L4 upon hybridization of the
`same blot with the C, probe is consistent with the above inter-
`pretation. DNA from this transformant contains a 9.6-kbp frag-
`ment corresponding to the wild-type Kmp gene (16) in addition
`
`Phenotype
`IgM, K(TNP)
`+ K(X63)
`K(TNP)
`
`IgG1, K
`
`No Ig
`
`Cell line
`speoa
`
`igm-10
`IR44L1
`
`X63Ag8
`XR19L4‘
`
`Sp2/0Ag14
`SR1.2
`SR40.1
`
`X63Ag8.653
`X653R1.1
`
`No Ig
`
`£ TNP/protein
`Protein A
`TNP
`A ratio
`2‘
`2°
`4
`
`<1
`23
`
`<1
`23
`
`<1
`2‘
`2
`
`<1
`2‘
`
`<1
`2‘
`
`<1
`<1
`
`<1
`2“
`2’
`
`<1
`2‘
`
`—
`4
`
`—
`<1 :8
`
`_
`4
`2
`
`—
`4
`
`As described in the text, the transformants lR44L1 and XR19L4 were ~
`derived by introducing the F/[Np gene alone or the [L1'Ny and K-mp genes
`together into the igm-10 and X63Ag'8 cell lines. Similarly, the cell lines
`SR1.2, SR40.1, and X653R1.1 were generated by transferring the amp ~
`+ K-mp vector DR-'I'III1'Np into Sp2/0Ag14 and X63Ag8.653. The indi-
`cated cell lines were grown to approximately 10° cells per ml,‘ and cul-
`ture supematants were assayed for IgM concentration (lysis titer on
`protein A-coupled erythrocytes) and TNP-specific hemolysis activity
`(lysis titer on TNP-coupled erythrocytes). Culture supernatants were A
`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 IgM. ‘
`
`to other fragments that correspond to the K chain genes en-
`dogenous to the recipient X63Ag8-cell line (23, 24).
`Assay of IgM.Function. We have tested the normal func-
`tioning of the IgM produced by the transformants by assaying
`its action in complement-dependent
`lysis of TNP-coupled
`erythrocytes (Table 1). The IgM concentration in the culture
`supematants of the indicated cell lines was measured. by the
`hemolysis of protein A-coupled erythrocytes in the presence of
`anti-IgM
`These results indicate that IgM made by IR44L1
`has normal activity with regard to TNP binding-and comple-
`ment activation. However, the transformant XR19L4 makes IgM
`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
`IgM, thus reducing TNP-specific hemolysis activity (7). To avoid
`this problem of the nonspecific myeloma K chain, the pq-Np +
`K1'Np vector pR-HL-mp was transferred into theanonproducer
`cell lines Sp2/0Agl4 (5) and X63Ag8.653 (4). The. IgM pro-
`duced by transfonnants-of these cell lines has normal activity
`for TNP-specific hemolysis (Table 1).
`
`DISCUSSION
`
`We and others have previously reported the expression of Ig
`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 p. and K-chains. The expres-
`sion of these genes was studied after the transferof the plas-
`mids into various cell lines derived from Ig-secreting plasma-
`cytomas or hybridomas. The transfer of these plasmids into these
`cells is usually (see below) sufficient to cause the production of
`pentameric IgM(K) that binds antigen (TNP) and activates com-
`plement‘—that is, these cell lines (X63Ag8, X63Ag8.653, igm-
`10, and Sp2/0Ag14) provide all of’ the machinery necessary for
`IgM production except the structural genes for the )1. and,K
`
`Sanofi/Regeneron Ex. 1040, pg 1024
`
`Mylan Ex. 1040, pg 1024
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`

`
`Immunology: Ochi et al.
`
`Proc. Natl. Acad. Sci. USA 80 (1983)
`
`6355
`
`chains. The capacity to provide this machinery is present de-
`spite the fact 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 IgM production and
`function. To date, the use of genetics for this purpose has been
`limited to the analysis of naturally occurring mutants that in-
`terfere with normal IgM processing and activity (7, 30). Al-
`though such mutans are useful as a starting point, in vitro mu-
`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 Fc receptor
`binding. Similarly, one can expect to define the features that
`are necessary for pentamer formation, glycosylation, and se-
`cretion.
`As is the case with other gene transfer systems, we have found
`that the various transformants produce quite different amounts
`of )1. and K chain, ranging from undetectable to approximately
`normal levels. In general, a linear relationship does not exist
`between the copy number of the transferred sequences and the
`level of Ig gene expression. Studies with transfer vectors pre-
`sumed to be replication incompetent indicate that the trans-
`ferred sequences integrate into ‘different sites in the host chro-
`mosomes,
`independent of the 'method of transfer (31-33).
`Therefore, the context of the transferred genes is different from
`normal and different in each recipient. 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 as RNA, a 2.7-
`kb RNA that appears to include the am exon. As we have been
`unable to detect a';1.,,, 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 XRIQL4
`and in several transformants derived from igk-14 and the K1-Np
`vector pR-TK1 (ref. 2; unpublished data) produce a single spe-
`cies of RNA that comigrates with authentic K-mp RNA.
`We expect that the variations. in the expression of the trans-
`ferred genes will not interfere with the usefiilness of this sys-
`tem in producing altered IgM for functional analysis. Further-
`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-
`cently reported the expression of cloned heavy chain genes in trans-
`formed lymphoid cells.
`
`We thank Nusrat Covindji and Catherine Filkin for expert technical
`assistance. This work was supported by grants from the Medical Re-
`search Council, the National Cancer Institute, the Arthritis Society, the
`Allstate Foundation, and Hoffmann—La Roche Ltd. A.O. was sup-
`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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