`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 OCHI*t, ROBERT G. HAWLEY*t, TERESA HAWLEY*t, MARC J. SHULMANtt, ANDRE' TRAUNECKER§,
`GEORGES KOHLER§, AND NOBUMICHI HOZUMI*t
`*Ontaro 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 IJ3 Canada; and §Basel Institute for Immunology, Grenzacherstrasse 487, Basel CH-4005, Switzerland
`Communicated by Niels KajJerne, 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-
`troduced into various plasmacytoma and hybridoma cell lines. The
`transfer of the gi 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 Ig 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 ,u 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 ,. 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 IgGl(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/OAgl4 is an Ig 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 yl and K chains of X63Ag8 as well as the (TNP
`specific) UTNp 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-
`ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
`
`ing the yl chain was isolated, and the Sp602 and Sp603 cell
`lines were derived from this yl nonproducer. The mutant cell
`line igm-10, derived from Sp602 (7), lacks the gene encoding
`/.TNP (8).
`Gene Transfer. The construction of pSV2-neo plasmid vec-
`tors carrying the genes for ATNp or KTNp or both is described
`in the text. The vectors were transfected into the rk-mk 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/OAgl4, 10`5 for igm-10, and 10-6 for
`X63Ag8.653.
`Analysis of 1g. As described previously (7), Ig 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 Schibler 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-TKI, where TK1 is inserted in the BamHI
`site of the vector pSV2-neo (2, 17). The /.TNP 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-
`tained from Sp6-718 after partial digestion with EcoRI and was
`inserted at the EcoRI site of the vectors pSV2-neo and pR-TK1.
`In these recombinants, designated pR-Sp6 and pR-HLTNp, re-
`Abbreviations: TNP, 2,4,6-trinitrophenyl; ELISA, enzyme-linked im-
`munoadsorbent assay; kbp, kilobase pair(s); SV40, simian virus 40; kb,
`kilobase(s).
`
`6351
`
`Genzyme Ex. 1035, pg 900
`
`
`
`1-1 el r_16352
`
`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-TKI-i.e., the direction of transcription of ATNP
`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 /TNp 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 KTNp gene (2).
`Expression of the tTNp gene of pR-Sp6 was assayed here in
`igm-10. The simultaneous production of both ITNp and KTNp
`chains from the vector pR-HLTNp is assayed in X63Ag8, the IgGl-
`producing plasmacytoma parent of the Sp6 hybridoma. In later
`experiments the pR-HLTNp vector was assayed in the non-
`producing cell lines Sp2/OAgl4 and X63Ag8.653. IgM pro-
`duction by the transformants is compared with Sp603, a sub-
`clone of the Sp6 hybridoma.
`Selection of IgM(oc)-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
`
`CL gBom HI
`
`EcoRI
`
`VHTNP I
`
`the antibiotic G418 (17). To transfer the Ig genes into the hy-
`bridoma 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 efficiency of G418-re-
`sistant colonies ranged between 10-4 and 10-6 per input hy-
`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 of TNP-coupled erythrocytes. In various
`experiments between 15% and 75% of the colonies were pos-
`itive in such tests.
`Analysis of ILTNP and KTNp Production. Colonies that were
`positive for TNP-specific IgM were cloned by limiting dilution
`and examined further. The transformant IR44L1, derived from
`the KTNp-positive cell line igm-10 and the uTNp vector pR-Sp6,
`makes about 25% of the normal (Sp603) amount of IgM, as
`measured by the TNP-dependent ELISA. The transformant
`XR19L4, derived from the cell line X63Ag8 and the kTNp +
`KTNp vector pR-HLqp, makes about 10% of the normal amount
`of IgM.
`To examine the TNp and KTNp 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 TNp and KTNp chains (Fig. 2, lane
`e). The XR19L4 transformant derived from X63Ag8 has two ad-
`ditional bands (Fig. 2, lane b), which comigrate with the tLTNp
`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-
`sumably because of a rearrangement in this K gene (see legend
`to Fig. 5). The IR44L1 transformant derived from igm-10 has
`one new band that comigrates with PLTNp (Fig. 2, lane d). As
`shown in Fig. 3, analysis of unreduced IgM by NaDodSO4/
`polyacrylamide gel electrophoresis indicates that the trans-
`formants make predominantly pentameric IgM [(pt2K2)5].
`RNA Production. To examine the RNAs expressed by the
`transferred uTNp and KTNp genes, cytoplasmic RNA from the
`transformants was fractionated by gel electrophoresis and probed
`
`a
`
`b
`
`c
`
`d
`
`e
`
`FL.
`
`EcoRI
`Structure of the pR-Sp6 and pR-HLmp plasmids. pR-Sp6
`FIG. 1.
`contains the functionally rearranged ptNp gene (416 kbp), which was
`inserted into the EcoRI site of pSV2-neo (see text). In addition to the
`mP gene, pRHIbp contains the functionally rearranged KTw gene
`(9.6 kbp) at the BamHI site (2). Ig genes are represented by heavy dark
`lines. The directions oftanscription ofthe Ig genes and the SV40 early
`region are indicated by arrows. The g and K exons are shown as fied
`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.
`
`K -
`TNP
`
`m.
`
`Analysis of heavy and light chains of secreted Ig. G418-
`FIG. 2.
`resistant transformant clones were biosynthetically radiolabeled with
`['4Clleucine 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.
`
`Genzyme Ex. 1035, pg 901
`
`
`
`Immunology: Ochi et al.
`
`Proc. Natl. Acad. Sci. USA 80 (1983)
`
`6353
`
`a
`
`b
`
`c
`
`d
`
`e
`
`ita KNP )5{
`2
`2
`
`Ut
`
`B"a
`
`A
`
`kb
`
`a
`
`b
`
`c
`
`d
`
`e
`
`_
`
`_
`
`_
`
`2.7-
`
`2.4-t U
`
`)UKTNP W
`
`Analysis of secreted (unreduced) Ig. The radiolabeled cul-
`FIG. 3.
`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-
`10; lane d, 1R44L1; and lane e, wild-type hybridoma Sp603. The mark-
`ers indicate the major forms of Sp603 1gM and X63Ag8 IgG1.
`
`with various ,u- and K-specific DNA sequences (Fig. 4). RNA
`heavy chain was detected with a probe from the CM4
`for the
`region. The transformants XR19L4 and IR44L1 have bands at
`both 2.7 and 2.4 kilobases (kb), whereas the parental hybridoma
`Sp6O3 has only one band at 2.4 kb (Fig. 4A). A genomic probe
`containing the A 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 (A,) and secreted (,) forms
`of the A chain, respectively (19-21). These results suggest that,
`whereas Sp6O3 makes RNA only for the As form, the trans-
`formants make RNAs for both p. and As. However, we have
`been unable to detect membrane IgM by staining with flu-
`orescent p-specific antibodies. The A, form has a longer poly-
`peptide chain than does the As form and consequently can be
`distinguished from ,s by its lower mobility in NaDodSO4/
`polyacrylamide gel electrophoresis. Therefore, we examined
`intracellular A& chains that were biosynthetically radiolabeled in
`the presence of tunicamycin; for each transformant we found
`only one ,u band, and this band comigrated with the ,u band of
`Sp6 (results not shown). These observations suggest that either
`the 2.7-kb RNA is not translated or that the A, 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 Sp6O3 and
`igm-10, the transformant XR19L4 was found to make a low
`amount of a 1.2-kb RNA that comigrated with authentic KTNp
`RNA (Fig. 4B).
`Structure of Transferred DNA. To analyze the organization
`of the transferred pR-Sp6 and pR-HLTNp plasmids in the trans-
`formed cell lines, BamHI-digested cell DNA was hybridized
`with probes specific for the u- and K-chain constant region gene
`segments. The CA1-2 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.
`
`B
`
`a
`
`b
`
`c
`
`d
`
`e
`
`i.2-
`
`.
`
`.
`
`Detection of /TNP and KTyp gene sequences in cytoplasmic
`FIG. 4.
`RNA from transformed cell lines. Lanes a, X63Ag8; lanes b, XR19L4;
`lanes c, igm-10; lanes d, ER44L1; 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 32P-labeled probe correspond-
`ing to the CQ4 exon. This probe was isolated from the cDNA clone
`pH76,.17 (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 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-HLTNp plasmids (Fig. 5). In addition, one (XR19L4) or two
`(IR44L1) extra fragments could be detected in the DNA from
`these cell lines. In parallel experiments, sequences indicative
`of unintegrated pR-TKI 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-
`ient cells.
`
`Genzyme Ex. 1035, pg 902
`
`
`
`Immunology: Ochi et al.
`
`Proc. Natl. Acad. Sci. USA-80 (1983)
`
`6354
`
`A
`
`i-
`
`b
`
`c
`
`d
`
`e f
`
`."..
`
`I
`
`kop
`
`16 -
`
`6.0-
`
`Table 1. Assay of functional IgM
`Hemolysis titer
`on erythrocytes
`coupled with
`Protein A
`TNP
`24
`26
`
`Cell line
`Sp6O3
`
`TNP/protein
`A ratio
`4
`
`Phenotype
`IgM, #c(TNP)
`+ ic(X63)
`#c(TNP)
`
`IgGl, K
`
`No Ig
`
`igm-10
`ER44L1
`
`X63Ag8
`XR19L4
`
`Sp2/OAgl4
`SR1.2
`SR40.1
`
`<1
`
`3
`
`<1
`23
`
`<1
`24
`2
`
`<1
`25
`
`<1
`<1
`
`<1
`26
`22
`
`-
`4
`
`-
`<1:8
`
`-
`4
`2
`
`B
`
`kbp
`
`a
`
`u
`
`c
`
`u
`
`e
`
`f
`
`9.6-
`
`69- *
`59-i:
`5.4-
`
`-, V-fo~
`
`--~~~~~~~~~~~~~~~~~~~~~~~~~~..
`-*00.
`
`........
`
`Detection ofpR-Sp6 and pR-HLTNP sequences in DNA from
`FIG. 5.
`transformed cell lines. Lanes a, X63Ag8; lanes b, XR19L4; lanes c, igm-
`10; lanes d, IR44L1, lanes e, Sp603; and lanes f, igm-10 with 5 equiv-
`alents of pR-Sp6. BamHI-digested DNA samples (20 Ag) were electro-
`phoresed through a 1% agarose gel at 2 V cm- 1for 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 CA1 and C,,2 exons. This probe was prepared by isolation of
`an appropriate fragment from a Xba I/Hindil digestion of a genomic
`clone of the ,-chain constant region gene segment. The bands corre-
`sponding to the A-chain gene-containing fragments generatedbyBamHI
`digestion of pR-Sp6 and pR-HLTNp are indicated. The two bands ob-
`served in lane e (11 and 14 kbp) correspond to the functionally rear-
`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-
`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 KTNp 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-
`bridoma. The 5.4-kbp band corresponds to the functionally rearranged
`X63Ag8 K gene and this band is not observed in the case ofigm-10 (lane
`c). Sizes were estimated by comparison toHindRI-digested A phage DNA.
`The pattern obtained for XR19L4 upon hybridization of the
`same blot with the CK probe is consistent with the above inter-
`pretation. DNA from this transformant contains a 9.6-kbp frag-
`ment corresponding to the wild-type KTNp gene (16) in addition
`
`No Ig
`
`X63Ag8.653
`<1
`<1
`24
`X653R1.1
`26
`4
`As described inthe text,-the transformantsIR44L1 and XR19L4-were
`derived by introducing the pTNp gene alone or the T
`and KTNp genes
`together into the igm-10 and X63Ag8 cell lines. Similarly, the cell lines
`SR1.2, SR40.1, and X653RL.1 were generatedby transferring the ATNp
`+ KTNp vector pR-HLrNp into Sp2/OAg14 and X63Ag8.653. The indi-
`cated cell lines were grown to approximately 106 cells per ml, and cul-
`ture supernatants were assayed for IgM concentration (ysis 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 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
`supernatants of the indicated cell lines was measured. by the
`hemolysis of protein-A-coupled erythrocytes-in the presence of
`anti-IgM (7). 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 ,UTNp +
`KTNp vector pR-HLTNp was transferred into the-nonproducer
`cell lines Sp2/OAgl4 (5) and X63Ag8.653 (4). The. IgM pro-
`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 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 A and K-chains. The expres-
`sion of these genes was studied after the transfer-of 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/OAgl4) provide all of the machinery necessary for
`IgM production except the structural genes for the p. and. K
`
`Genzyme Ex. 1035, pg 903
`
`
`
`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 mutants 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 g 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 p, RNA, a 2.7-
`kb RNA that appears to include the P, exon. As we have been
`unable to detect aI.m 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-
`cies of RNA that comigrates with authentic KTNp RNA.
`We expect that the variations in the expression of the trans-
`ferred genes will not interfere with the usefulness 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 Govindji 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.
`Davies, D. & Metzger, H. (1983)Annu. Rev.-Immunol. 1, 87-117.
`1.
`Ochi, A.,.Hawley, R. G., Shulman, M. J. &-Hozumi, N. (1983)
`2.
`Nature (London) 302, 340-342.
`Kohler, G.& Milstein, C. (1975) Nature (London) 256, 495-497.
`Kearney, J., Radbruch, A., Liesegang, B. & Rajewsky, K. (1979)
`J. Immunol.;123, 1548-1550.
`
`3.
`4.
`
`5.
`
`6.
`7.
`8.
`
`9.
`
`10.
`11.
`
`12.
`13.
`
`14.
`
`15.
`
`16.
`
`17.
`18.
`
`19.
`
`20.
`
`21.
`
`22.
`
`23.
`
`24.
`
`25.
`26.
`
`27.
`
`28.
`
`29.
`
`30.
`
`31.
`
`32.
`
`33.
`
`34.
`
`35.
`
`36.
`
`37.
`
`38.
`
`39.
`
`40.
`
`41.
`
`Shulman, M., Wilde, C. & Kohler, G. (1978) Nature (London) 276,
`269-270.
`Kohler, G. & Milstein, C. (1976) Eur. J. Immunol. 6, 511-519.
`Kbhler, G. & Shulman, M. (1980) Eur. J. Immunol. 10, 467-476.
`Kohier, G., Potash, M. J., Lehrach, H. G. & Shulman, M. J. (1982)
`EMBOJ. 1, 555-563.
`Schibler, U., Marcu, K. B. & Perry, R. P. (1978) Cell 15, 1495-
`1509.
`Thomas, P: S. (1980) Proc. Natl. Acad. Sci. USA 77, 5201-5205.
`Gross-Bellard, M., Dudet, P. & Chambon, P. (1973) Eur. J.
`Biochem. 36, 32-38.
`Southern, E. M. (1975)J. Mol. Biol. 97, 503-517.
`Rigby, P. W. J., Dieckmann, M., Rhodes, C. & Berg, P. (1977)J.
`Mol. Biol. 113, 237-251.
`Hozumi, N., Hawley, R. G. & Murialdo, H. (1981) Gene 13, 163-
`172.
`Hozumi, N., Wu, G. E., Murialdo, H., Roberts, L., Vetter, D.,
`Fife, W. L., Whiteley, M. & SadowskiP. (1981) Proc. Nati. Acad.
`Sci. USA 78, 7019-7023.
`Hawley, R. G., Shulman, M. J., Murialdo, H., Gibson, D. M. &
`Hozumi, N. (1982) Proc. Natl. Acad. Sci. USA-79, 7425-7429.
`Southern, P. J. & Berg, P. (1982)J. Mol. Appl. Genet. 1, 327-341.
`Gough, N. M., Kemp, D. J., Tyler, B. M., Adams, J. M. & Cory,
`S. (1980) Proc. Natl. Acad. Sci. USA 77, 554-558.
`Alt, F. W., Bothwell, A. L. M., Knapp, M., Siden, E., Mather,
`E., Koshland, M. & Baltimore, D. (1980) Cell20, 293-301.
`Rogers, J., Early, P., Carter, C., Calame, K., Bond, M., Hood,
`L. & Wall, R. (1980) Cell 20, 303-312.
`Early, P., Rogers, J., Davis, M., Calame, K., Bond, M., Wall, R.
`& Hood, L. (1980) Cell 20, 313-319.
`Wall, R., Gilmore-Hebert, M., Higuchi,. R., Komaromy, M.,
`Paddock, G., Strommer, J. & Salser, W. (1978) Nucleic Acids Res.
`5, 3113-3128.
`Storb, U., Arp, B. & Wilson, R. (1980) Nucleic Acids Res. 8, 4681-
`4687.
`Walfield, A. M., Storb, U., Selsing, E. &-Zentgraf, H. (1980) Nu-
`cleic Acids Res. 8, 4689-4707.
`Hirt, B. (1967)J. Mol. Biol. 26, 365-369.
`Rice, D. & Baltimore, D. (1982) Proc. Natl. Acad. Sci. USA 79,
`7862-7865.
`Oi, V. T., Morrison, S. L., Herzenberg, L. A. & Berg, P. (1983)
`Proc. Natl. Acad. Sci. USA 80, 825-829.
`Falkner, F. G. & Zachau, H. G. (1982) Nature (London) 298, 286-
`288.
`Picard, D. & Schaffner, W. (1983) Proc. Natl. Acad. Sci. USA 80,
`417-421.
`Shulman, M. J., Heusser, C., Filkin, C. & Kohler, G. (1982) Mol.
`Cell. Biol. 2, 1033-1044.
`Robins, D. M., Ripley, S., Henderson, A. S. & Axel, R. (1981)
`Cell 23, 29-39.
`de Saint Vincent, B. R., Delbruk, S., Eckhart, W., Meinkoth, J.,
`Vitto, L. & Wahl, G. (1981) Cell 27, 267-277.
`Folger, K. R., Wong, E. A., Wahl, G. & Capecchi, M. R. (1982)
`Mol. Cell. Biol. 2, 1372-1387.
`Razzaque, A., Mizusawa, H. & Seidman, M. M. (1983) Proc. Natl.
`Acad. Sci. USA 80, 3010-3014.
`Calos, M. P., Lebkowski, J. S. & Botchan, M. R. (1983) Proc. Natl.
`Acad. Sci. USA 80, 3015-3019.
`Kemp, D. J., Harris, A. W. & Adams, J. M. (1980) Proc. Natl. Acad.
`Sci. USA 77, 7400-7404.
`Alt, F. W., Rosenberg, N., Enea, V., Siden, E. & Baltimore, D.
`(1982) Mol. Cell. Biol. 2, 386-400.
`Clarke, C., Berenson, J., Goverman, J., Boyer, P. D., Crews. S.,
`Siu, G. & Calame, K. (1982) Nucleic Acids Res. 10, 7731-7749.
`Nelson, K. J., Haimovich, J. & Perry, R. P. (1983) Mol. Cell. Biol.
`3, 1317-1332.
`Gillies, S. D., Morrison, S. L., Oi, V. T. & Tonegawa, S. (1983)
`Cell 33, 717-728.
`Neuberger, M. S. (1983) EMBOJ. 2, 1373-1378.
`
`Genzyme Ex. 1035, pg 904