Journal of Immunological Methods, 130 (1990) 49-55

49

Elsevier JIM05583

Selective cloning of B cell hybridoma-specific rearranged immunoglobulin gene loci using the polymerase chain reaction M a r k u s Zwickl, D a n i e l a Z a n i n e t t a , G a r y K. M c M a s t e r a n d N o r m a n H a r d m a n Department of Molecular Biology, Biotechnology Section, Ciba-Geigy A G, CH-4002 Basel, Switzerland

(Received 27 November 1989, revised received 5 February 1990, accepted 12 February 1990)

The use of conventional DNA cloning procedures to obtain productively rearranged Ig genes from B cell hybridomas for structure/function analysis of immunoglobulins is tedious and time-consuming. Here we describe a procedure based on PCR which permits rapid, selective isolation of DNA segments containing individual hybridoma-specific Ig gene rearrangements. The method, an adaptation of the so-called 'inverted PCR' technique (IPCR), can be applied most efficiently to specific genes where a preliminary restriction map is available from Southern blot analysis of the hybridoma genomic DNA. To achieve amplification of a given rearranged Ig locus, small amounts of total hybridoma D N A are digested to completion with a chosen restriction endonuclease and the fragments circularised by DNA ligase. Cleavage of the DNA circles using a second restriction enzyme, chosen specifically to cut 3' to a rearranged V-(D)-J exon, leads to linear DNA segments where the rearranged gene is now flanked by segments of known nucleotide sequence derived originally from the 3' region of the Ig H or L chain gene locus. This permits the selection of oligonucleotides that provide convergent primers for specific amplification of DNA segments containing the required gene rearrangement. Amplified DNA fragments can be cloned and rapidly characterised by sequence analysis. Key words: Polymerasechain reaction; lmmunoglobulingene locus; Cloning

Introduction The specificity and function of B and T lymphocytes is dependent upon the presence of specific antigen receptors on their cell surface. Structural studies of these receptors, the im-

Correspondence to: N. Hardman, Biotechnology Section, K-681.4.43, Ciba-GeigyAG, CH-A002Basel, Switzerland(Tel.: 061 696 70 61). Abbreviations: kb, kilobase (pairs); PCR, polymerasechain reaction; IPCR, inverted polymerasechain reaction.

munoglobulins on B iymphocytes and T cell receptors on T lymphocytes, provides the basis for understanding how the molecules participate in antigen recognition. Such studies are aided by rapid procedures for isolating and characterising immunoglobulin and T cell receptor cDNAs and their corresponding genes. These sequences can be obtained by application of conventional DNA cloning and library screening procedures, but this is a tedious and time-consuming process. A recent innovation has made use of the polymerase chain reaction (PCR) (Saiki et al., 1988; White et al., 1989) for selective amplification of receptor

0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

50 cDNAs from heterogeneous cellular mRNA preparations after generating first-strand cDNA by reverse transcription, thus avoiding lengthy cloning and sequencing steps (Lacy et al., 1989; Loh et al., 1989; Orlandi et al., 1989). In one adaptation, so far applied to immunoglobulin H and L chain cDNAs from hybridoma cells, consensus oligonucleotide primers corresponding to either end of the Ig variable region have been used (Chiang et al., 1989; Orlandi et al., 1989). An alternative and more general method described by Loh et al. (1989), referred to as the 'anchored polymerase chain reaction' has been applied to T cell receptors. For this approach a homopolymer dG tail is added to the 3' end of the first-strand cDNA to provide a template for reverse priming with poly-dC in the PCR reaction. A second oligonucleotide primer, corresponding to an internal site in the constant region of the receptor cDNA coding sequence, permits selective amplification of the appropriate cDNA sequences. Although these procedures have been adapted successfully for receptor cDNA isolation, two main problems have precluded the application of PCR for isolating receptor genes from genomic DNA. The first problem arises since amplification requires two complementary oligonucleotide primers flanking the gene segment of interest. Such information is not normally available for antigen receptor genes since the sequences of the V regions are often unknown and usually heterogeneous. The second problem stems from practical limitations of the PCR technique; small DNA fragments such as cDNAs can be amplified efficiently, but large segments of genomic DNA amplify inefficiently or not at all. These problems have been overcome in the present study, where a variation of the PCR method, referred to as 'inverted PCR' (IPCR) (Ochman et al., 1988; Triglia et al., 1988), has been used for specific amplification of genomic DNA segments containing functionally rearranged H and L chain V regions of a B cell hybridoma. In order that the IPCR procedure can be generally used to amplify Ig gene loci it has been necessary to modify the composition of the buffer used to perform PCR. The buffer system described permits the amplification of comparatively long genomic DNA fragments, in excess of 5-6 kb.

Materials

and

methods

Preparation of DNA for amplification Genomic DNA was isolated by the method of Nielson et al. (1985) from the murine hybridoma 4-15 which produces a neutralising MAb directed against human respiratory syncytial virus (Gimenez et al., 1986). The hybridoma was obtained by fusion between spleen cells of immunised BALB/c mice and the myeloma fusion partner P3-X63.Ag8.653. DNA was digested using restriction endonucleases according to the manufacturers' instructions. Where indicated restriction fragments were size-fractionated by electrophoresis on 0.6% agarose gels (Ultrapure grade, Sigma, Glattburg, Switzerland) and DNA recovered by electroelution/ethanol precipitation. Ligation of DNA fragments was carried out at 14°C for 15 h in buffer containing 10 mM MgCI 2, 20 mM dithiothreitol, 1 mM ATP, 50 mM Tris-HCI, pH 7.8 under conditions favouring circularisation (DNA concentration < 3/~g/ml). Ligated DNA was then purified by phenol extraction and ethanol precipitation, and after recovery was linearised using a second restriction endonuclease. The position of the second DNA restriction site permitted the generation of linear molecules suitable for amplification using PCR (see results section).

DNA amplification and Southern blot analysis The following oligonucleotide primers were used for DNA amplification. For murine H chain gene sequences: CH2, 5 'dAAGGCTCTGAGATCCCTAGACAG3' DH3, 5'dAATGGGAGTGAGGCTCTCTCATA3' For murine L chain gene sequences: AL2, 5'dCTGACACTGTATGCCACGTCAAC3' J2, 5'dCCAGC'I'TGGTCCCCCCTCCG3' BL1, 5 'dGGTCTGACTGCAGGTAGCGTGGT3' BL2,

5 "dCCCAGAGGATGAAACAGCGCAGA3' For Southern blot characterisation of amplified H and L chain gene segments the following J minigene oligonucleotides were used as probes. For H chain:

51

JH3, 5 ' d C T G C A G A G A C A G T G A C C A G A 3 ' - For L chain: J2 (see above) J5, 5 ' d C C A G C T r G G T C C C A G C A C C G 3 ' Total genomic D N A (500 ng) or sizefractionated D N A (200 ng) was amplified in a total volume of 100 #1. The amplification buffer contained 10% ( v / v ) dimethylsulphoxide (Merck, Ziirich), 50 mM KCI, 1.5 m m MgCI 2, 0.2 m M d N T P ( N = G , C, A or T), 0.05% Tween 20 (Merck, Ziirich), 0.05% NP-40 (Merck, Ziirich), 10 m M fl-mercaptoethanol, 50 p m o l of each oligonucleotide primer and 2.5 U of Taq D N A polymerase (Perkin Elmer Cetus, Kiisnacht, Switzerland). Samples were overlaid with 100/~1 of paraffin oil and subjected to 30-40 cycles of amplification using the Hybaid Intelligent Heating Block (AMS Biotechnology, Ziirich). D N A denaturation was achieved by heating to 93 ° C. Samples were immediately cooled and annealed at 63-65 ° C for 0.2 rain. D N A synthesis was carried out at 71°C for 2 min. After the last amplification step samples were incubated for an additional 5 - 1 0 min at 71°C to allow completion of D N A chains. One-tenth of the reaction mixture was analysed on 0.8% agarose gels stained with 0.5 /zg/ml ethidium bromide in order to visualise the amplified D N A products. Southern blot analysis of hybridoma genomic D N A was carried out as previously described by H a r d m a n et al. (1989). PCR amplification products were analysed by Southern blot hybridisation using murine Ig J minigene oligonucleotide probes using conditions described previously by Rooney et al. (1988).

Results

Principle of the IPCR procedure Fig. 1 illustrates how the I P C R method can be applied to amplify selectively rearranged Ig V regions from B cell hybridoma genomic DNA. The positions of D N A restriction sites located 3' of the murine H and L chain Ig J regions can be predicted from available nucleotide sequence information. Restriction analysis and Southern blotting are used to identify hybridoma D N A fragments containing hybridoma-specific rearranged

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Fig. 1. Principle of the IPCR method as applied to the isolation of rearranged lg gene loci. The boxed regions indicate the positions of the coding sequences for the leader peptide (L). V region (V) and J segment (J) of a rearranged H or L chain Ig gene. A and B indicate the positions of hypothetical DNA restriction sites surrounding the gene locus that are useful for 1PCR, deduced from Southern blot analysis of hybridoma genomic DNA. x and y' represent PCR primer oligonucleotides complementary to the different DNA strands located 3' to the rearranged Ig gene. a: genomic DNA is cleaved with enzyme A and ligated under conditions favouring DNA circle formation, b: the ligation products are digested using enzyme B, generating a linear molecule in which the rearranged lg gene is flanked by sequences complementary to the now convergent PCR primers x and y', providing an appropriate orientation for amplification, as in (c). lg gene loci using J region hybridisation probes, and an enzyme (A) is selected which provides restriction fragments of convenient size for gene amplification. The fragments can be large enough to include the complete rearranged V region and additional sequences as required, for example leader peptide exons and promoter elements. Following digestion, D N A is ligated under conditions favouring circularisation. Amongst the ligation products are circles containing the rearranged lg V region. D N A is then digested with a second enzyme ( B ) selected on the basis of Southern blotting data to cut circles containing Ig gene segments in the region of known sequence, generating linear fragments of the form shown in Fig. 1. Oiigonucleotides (x and y ' ) corresponding to sequences on either side of restriction site B located 3' to the rearranged Ig locus are synthesised for

52 probe

use as PCR primers, allowing specific amplification of a DNA segment containing the required V region.

Amplification

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of hybridoma-specific lg gene re-

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arrangements

probe CH2

To illustrate the utility of the procedure it was used in this study for selective amplification of the functionally rearranged Ig loci of the murine B cell hybridoma 4-15, which secretes a neutralising antibody directed against human respiratory syncytial virus (Gimenez et al., 1986). H and L chain J region hybridisation probes were used to detect immunoglobulin gene rearrangements by Southern blot analysis using a variety of restriction endonucleases. The results, summarised in Table I, reveal the presence of two hybridoma-specific L chain gene rearrangements, and two H chain gene rearrangements. The data were used to devise a strategy for selective amplification of these Ig gene loci using specific oligonucleotides as primers for IPCR (Fig. 2). One of the hybridoma-specific L chain gene rearrangements was selectively amplified as a 1.3 kb fragment using XbaI (enzyme A), Bglll (enzyme B) and the IPCR primers AL2 and BLI (Fig. 3a, lane 1). The second L chain gene rearrangement was amplified using BamHl (enzyme A), XbaI (enzyme B) and IPCR primers BL2 and J2 (Fig. 3a, lane 2). Both of the hy-

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Fig. 2. Restriction maps of the murine Ig gene loci in the germ-line configuration, a: L chain locus; and b: H chain locus. Numbered black boxes refer to J minigene exons, C, to the L chain constant region exon, and filled circles labelled e L and e H to enhancer elements. Triangles indicate approximate positions and directionality of PCR primers referred to in the text. Thick bars show the origin of H and L chain J region DNA probes used for genomic Southern blot analysis (Table I). Restriction sites: B, BamHI; Bg, Bglll; E, EcoRl; H, Hindlll; M, Mstll; P, Pstl; X, XbaI.

bridoma-specific H chain genes were amplified using EcoRI (enzyme A), XbaI (enzyme B) and H chain IPCR primers DH3 and CH2 (Fig. 3a, lanes 3 and 4). In this case only the smaller of the two PCR products (2.7 kb, lane 3) was generally recovered starting with total genomic DNA. Size fractionation of genomic DNA was required to eliminate this gene from the fragment pool in order to achieve amplification of the larger (4.5 kb) gene fragment (lane 4).

TABLE I S O U T H E R N BLOT ANALYSIS OF lg G E N E R E A R R A N G E M E N T S IN G E N O M I C DNA OF M U R I N E HYBRIDOMA CELL LINE 4-15 The positions of H and L chain J region DNA probes, and convenient DNA restriction sites, are shown in Fig. 2. H, 4-15 hybridoma cell DNA; P, parental myeloma-cell (P3-X63.Ag8.653) DNA; G, germ-line ( B A L B / c kidney) DNA configuration. Figures in brackets refer to DNA fragment sizes expected for Ig loci in the germ-line configuration. Asterisks denote DNA bands containing hybridoma-specific Ig gene rearrangements. Source of DNA probe

DNA fragment size (kb) detected using restriction enzyme indicated

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H chain

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53

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Fig. 3. Analysis of amplified Ig gene segments of the murine hybridoma 4-15. a: 0.6% agarose gel of products of IPCR amplification. Lane 1 and 5, L chain amplification product obtained with Xbal(enzyme A)/Bglll(enzyme B) using primers AL2 and BL1; lanes 2 and 6, L chain amplification product obtained with BamHI(enzymeA)/Xbal(enzyme B) using primers J2 and BL2; Lane 3 and 4, H chain amplification products obtained with EcoRl(enzyme A)/Xbal(enzyme B) using primers CH2 and DH3. DNA in lane 3 was amplified from total, unfractionated genomic DNA, whereas DNA in lane 4 was amplified from a size-fractionated pool of fragments of 4.2-6.0 kb obtained by agarose gel electrophoresis, b: Southern blot analysis of the same gels using the oligonucleotide probes indicated. M, radioactively labelled marker DNA fragments (mixture of Hindlll and Hindlll/EcoRl bacteriophage h DNA fragments). Figures in b refer to approximate sizes of amplification products in kb.

A nalysis of amplification products Fig. 4 summarises the structures of the amplified gene fragments and the positions of the D N A restriction sites and primers used for amplification using IPCR. Nucleotide sequencing was carried out to distinguish functional from non-functional V-(D)-J rearrangements. F r o m this analysis it could be demonstrated that the 1.3 kb and 5.5 kb L chain gene segments are rearranged at J5 and J2, respectively ( L l f and L2n; Fig. 4 a ) . Both the 2.7 kb and 4.5 kb H chain gene segments are rearranged at JH3 (H3f and H2n; Fig. 4b). Results showing hybridisation of the amplified fragments with the relevant J minigene oligonucleotide

probes are presented in Fig. 3b. L l f and H3f genes contain in-frame V-J and V-D-J rearrangements and encode the functional H and L chains of the 4-15 h y b r i d o m a ( H a r d m a n et al., u n p u b lished results) whereas L2n and H2n encode outof-frame, non-functional Ig gene rearrangements.

Discussion The I P C R m e t h o d has been used in earlier studies by O c h m a n et al. (1988) and Triglia et al. (1988) for the amplification of relatively small genomic D N A fragments up to 1 - 2 kb in length.

54 a. L - c h a i n

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