Communicated by Andre Lwoff, February 26, 1987 (receivedfor review February 6, 1987). ABSTRACT ..... We thank C. Fabre for excellent secretarial assistance.
Proc. Nat!. Acad. Sci. USA Vol. 84, pp. 4229-4233, June 1987 Immunology
Rearrangement and expression of rabbit immunoglobulin K light chain gene in transgenic mice (recombination sequence/allelic exclusion/spleen/thymus)
M. GOODHARDT*, P. CAVELIER*, M. A. AKIMENKO*, G. LUTFALLA*, C. BABINETt, AND F. ROUGEON** *Unitd de Gdndtique et Biochimie du Ddveloppement, and tGdndtique des Mammif~res, Centre National de la Recherche Scientifique, Unitd Associed 361, D6partement d'Immunologie, Institut Pasteur, 25, rue du Dr Roux, 75724 Paris Cddex 15, France
Communicated by Andre Lwoff, February 26, 1987 (received for review February 6, 1987)
To determine whether a foreign unreABSTRACT arranged immunoglobulin gene can be functionally rearranged and expressed in vivo, a rabbit b9 K light chain gene construct containing a single germ-line K chain variable (V) region gene (V1'), the five K chain joining (J) segments (J.K), and the K chain constant (C) region germ-line gene (C,,) was introduced into fertilized mouse eggs. Eleven transgenic mice carrying 1-30 copies of the rabbit K gene construct were obtained. Rearrangement of the transgene due to Ve-hJ recombination was observed in the spleen of all the mice lines analyzed. Only the JKi and J,2 segments, which have canonical sequences for rearrangement and high-level expression, are utilized in assembly of the rabbit gene. VK-JKi and Ve-Ja joining was also observed in the thymus but not in nonlymphoid tissue. Polyadenylylated rabbit K chain transcripts of 1.2 kilobases were found in the spleen of the transgenic mice. The level of transcription was low despite a high level of rearrangement. Three transgenic mice lines secreted K light chains encoded by the foreign rabbit gene. Serum rabbit K chains were associated with mouse , and Vi heavy chains. However, hybrid antibody molecules containing both rabbit and mouse K light chains were also found in the serum of these animals. These results suggest that, although the rabbit K chain gene construct contains the necessary sequences for gene assembly, sequences implicated in stage- and tissue-specific regulation of Kc chain gene rearrangement are either missing or not recognized by mouse lymphoid cells.
are site-specific and appear to be mediated by highly conserved recognition sequences located at the 3' or 5' border of all component gene segments (1). A typical recognition sequence consists of a palindromic heptamer that directly abuts the coding sequence and an A/T-rich nonamer, separated from the heptamer by a nonconserved spacer region of 12 or 23 base pairs (bp). These recombination sequences have been highly conserved for immunoglobulin genes throughout vertebrate evolution (11, 12) and are very similar to the sequences flanking the component gene segments involved in assembly of T-cell antigen receptor V regions (see ref. 13). More direct evidence that these sequences are the substrate for recombination enzymes has come from experiments showing that recombinant plasmids containing immunoglobulin or T-cell receptor gene segments with their flanking sequences can recombine when transfected into Abelson murine leukemia virus-transformed pre-B-cells (14-17). These in vitro transfection studies have suggested furthermore that transcription may play a direct role in the regulation of immunoglobulin gene recombination (16, 17). Recently, several groups have shown that functionally rearranged immunoglobulin genes introduced into the germ line of mice are correctly expressed and can alter the expression of the endogenous immunoglobulin repertoire (18-24). An unresolved question is whether an exogenous unrearranged immunoglobulin gene can be functionally rearranged and expressed when introduced outside its normal chromosomal location and in competition with the endogenous genes. To address this question, we have produced transgenic mice by microinjection of an unrearranged rabbit K light chain gene construct containing a single VK segment linked to a DNA fragment encompassing the germ-line JKe-CKb9 allele. The use of a heterologous rabbit gene provides a simple means of following the integration of the foreign gene and of distinguishing between expression of the introduced and endogenous K chain genes. Moreover, the rabbit b9 JK cluster contains five J elements but only two of these have the required sequences for rearrangement and high-level expression (25). The results of this study show that the unrearranged rabbit K chain gene is correctly assembled and expressed in the transgenic mice; however, rearrangement of the rabbit gene no longer appears to be under strict regulatory control.
Clonal expression of the antibody repertoire results from the ordered recombination of germ-line DNA segments coding for immunoglobulin heavy and light chain variable (V) regions (VH and VL, respectively; for reviews, see refs. 1 and 2). Assembly of immunoglobulin genes occurs during the early stages of B-cell development (3, 4), beginning with the formation of a functional VH gene from its component VH, diversity (D), and joining (JH) segments (by sequential joining of D to JH, then VH to D-JH). In-phase joining of VH-D-JH leads to the synthesis of cytoplasmic A heavy chains, which appears to signal the end of further heavy chain rearrangement and the start oflight chain assembly (5). Genes encoding K light chains are formed by joining one of several hundred germ-line V, segments to one offour or five JK segments lying several kilobases upstream of a single K chain constant (C) region gene (CK) (6-9). As for heavy chains, functional assembly of light chains occurs on only one chromosome (see ref. 10). Therefore, this method of gene assembly contributes to the diversity of the antibody repertoire by its combinatorial potential, while its strict regulation ensures the monospecificity of each B cell. Sequence analyses of germ-line gene segments and rearranged V genes have shown that the recombination events
MATERIALS AND METHODS Plasmid Construction and Microinjection. An 8.1-kilobase (kb) EcoRI-Sac I fragment of the recombinant phage X104 containing the rabbit b9 germ-line JK region and CK gene (26) was first introduced into the polylinker of pSP64 and then transferred into the EcoRI and Sal I sites of pBR322. The unrearranged rabbit K chain gene construct (pR plasmid) was
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Abbreviations: C, constant; J, joining; V, variable; D, diversity; VH, JH, and CH, heavy chain V, J, and C segment. *To whom reprint requests should be addressed.
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created by inserting in the EcoRI site a 2. 1-kb EcoRI fragment containing a germ-line VK gene (VK18a) isolated from a rabbit liver genomic library (27). The pR plasmid was linearized by cutting with Sal I and Sca I, and the 10.7-kb fragment containing 700 bp of rabbit sequences 5' of the VK gene and 600 bp 3' of the C,. gene (Fig. 1) was isolated on a 1% agarose gel. A few hundred copies of the linearized plasmid were injected into the pronucleus of fertilized eggs obtained from C57BL/6 x SJL/J hybrid female mice mated with identical hybrid males as described by Brinster et al. (28). Eggs that survived microinjection were implanted into the oviducts of pseudo-pregnant C57BL/6 x CBA foster females. F1 transgenic mice were obtained from backcross of founder (FO) pR mice with C57BL/6. DNA Analysis. DNA was prepared from tail biopsies or other organs as described (28, 29). Integration of the rabbit K chain gene was determined by Southern blot analysis of tail DNA using the JO5 or C,. probes described in the legend to Fig. 1. To detect rearrangement of the rabbit K chain gene, 20 ,ug of high molecular weight DNA was digested with Pvu II, fractionated by electrophoresis on 0.7% agarose gel, and transferred to Zeta-bind (Cuno) or Hybond (Amersham) nylon membranes. The membranes were hybridized with the probes indicated in Fig. 1 as described (29). Probes were radiolabeled with 32P to a specific activity of 1-3 x 109 cpm/Ag by the random priming method of Feinberg and Vogelstein (30) using random sequence hexanucleotides (Pharmacia). RNA Analysis. RNA was prepared from mouse and W rabbit spleen as described (31), except that 4 M guanidine thiocyanate was used instead of 6 M urea. For blot-hybridization experiments, RNA was electrophoresed on 1.1% agarose/6.6% formaldehyde gels transferred to nitrocellulose membranes and hybridized as described (32) with radiolabeled probes shown in Fig. 1. For the detection of mouse K chain transcripts, a 3-kb HindIII-BamHI fragment, which contains the mouse CK gene (6), was used as probe. Antibodies and Radioimmunoassays. Rabbit anti-b9 K chain antiserum, obtained by injection of purified immunoglobulins from a homozygous b9/b9 a2a2 rabbit into a b4/b4 a2a2 rabbit, was provided by P. A. Cazenave. Specific anti-b9 K chain antibodies (anti-b9 antibodies) were purified on a b9/b9 Ig-sepharose AH column. The specificity of this reagent was tested by RIA. All other polyclonal and monoclonal antibodies were provided by D. Primi and have been described (33). Direct binding experiments were carried out on plastic microtiter wells precoated with 20 ug of purified polyclonal anti-b9 antibodies per ml. Sera dilutions were incubated in the wells overnight at 4°C. After extensive washing, bound rabbit b9 immunoglobulin was revealed by incubation with "25I-labeled rabbit anti-b9 antibodies. Wells then were washed and assayed for radioactivity. Similarly, mouse
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heavy and light chains associated with the rabbit b9 K chains were detected by incubation with '25I-labeled polyclonal rabbit anti-mouse a chain, rabbit anti-mouse yl chain, or monoclonal rat anti-mouse K chain (139-52-1) antibodies.
RESULTS Rearrangement of Rabbit Kc Chain Gene in Mouse Spleen. Eleven transgenic mice were obtained that, as estimated from Southern blot analysis, had integrated 1-30 copies of the unrearranged rabbit K gene construct (Table 1). The rabbit germ-line JK cluster lies within a single Pvu II fragment of 5.5 kb. Since Pvu II also cleaves the rabbit VK gene used here in the leader sequence, VK-JK rearrangements should generate novel Pvu II fragments of smaller size (Fig. 1). Tail and spleen DNA from transgenic mice were digested with Pvu II and subjected to Southern blot hybridization analysis using a rabbitJ,5 probe (Fig. 1). A single 5.5-kb band corresponding to the unrearranged JK region was observed in the tail samples. Additional Pvu II fragments of 4.0 and 3.6 kb could be detected in spleen DNA from most of the transgenic mice obtained (Fig. 2, Table 1). These bands correspond to the size expected for V_-JK1 and VK_-JK rearrangement, respectively. Both the 4.0- and 3.6-kb bands hybridized to a VK probe, whereas only the higher band hybridized with a probe from the JK1-JK2 intersegment, and neither band hybridized to a 5' JK probe (data not shown; see Fig. 1 for probes). These results indicate that the rearranged fragments were generated by joining of the VK gene to the JK1 and J,2 segments and deletion of the intervening region. As estimated from densitometric scanning of the autoradiograms, the intensity of the rearranged JK bands for the foreign rabbit K chain gene is similar to that observed in b4 rabbit spleen. This would suggest that rearrangement of the rabbit K chain gene occurs in a large proportion of the mouse spleen cells. Furthermore, the frequency of rearrangement appears to be independent of the number of copies integrated (Fig. 2, Table 1). The Rabbit K Chain Gene Is Also Rearranged in Mouse Thymus. To further examine the tissue specificity of the rearrangement of the foreign K chain gene, Southern blot analysis of Pvu II digests of DNA from a number of different organs was performed. The results obtained for the F1 offspring of three independent transgenic mice lines (pR14, -16, and -19) are shown in Fig. 3. No rearrangement could be detected in nonlymphoid tissue such as heart, lung, liver, kidney, or brain. However, rearranged bands of 4.0 and 3.6 kb were observed in the thymus as well as in spleen for all three lines. The Foreign K Chain Gene Is Transcribed. Total and poly(A)+ RNA from the spleen of transgenic and control mice were subjected to blot-hybridization analysis using rabbit 1Kb
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FIG. 1. Physical map of linear pR DNA used for microinjection. Rabbit genomic sequences are depicted as a thick line (VK sequences) or thin line (Je-CK sequences). Closed bars represent coding sequences. pBR322 sequences are shown as a dashed line. E, Enhancer. The probes used are as follows: VK = 90-bp Pvu II-Pst I genomic fragment; 5' JK = 100-bp Aat I-Acc I fragment; J41_2 = 150-bp Sau3A fragment; JKS = 170-bp Hae III fragment; Int = 550-bp Alu I fragment; and CK = 320-bp Ava I-Kpn I fragment. Arrows indicate Pvu II (P) sites. Restriction enzymes used in the construct are: S, Sca I; E, EcoRI; and Sa, Sal I.
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Proc. Nati. Acad. Sci. USA 84 (1987)
Table 1. Analysis of transgenic mouse lines Rabbit K chain pR DNA Rearrangement Protein Copy RNA in spleen in serum Mouse no. in spleen + + + pR3 30 + + + pR4 7 +*pR5 12 +* 5 pR8 + ND pR14 10 + + + 25 pR16 1 ND ND pR17 3 ND ND pR18 + + 20 pR19 ND ND 1 pR20 + + pR25 1 The number of copies of pR DNA integrated per diploid genome of FO transgenic mice was estimated by Southern blot analysis using pR plasmid diluted to single copy in mouse genomic DNA as control. Rearrangement of the exogenous gene was determined by Southern blot analysis as depicted in Figs. 2 and 3. Expression of b9 rabbit K chain transcripts in spleen and the presence ofb9 K chain protein in the serum were determined by blot-hybridization analysis and RIA as shown in Figs. 4 and 5. ND, not determined; -, absent; +, present. *DNA was prepared from B cells isolated from the spleen of pR5 and pR8 as described (33).
CKb9 DNA as probe (Fig. 4A). Rabbit transcripts were detected in spleen from the transgenic mice, whereas the rabbit probe did not hybridize with spleen RNA from control mice that had not integrated the foreign rabbit gene (Fig. 4A). The transcripts correspond in size to K chain mRNA from rabbit spleen. Moreover, they hybridize to both ',, and C,. probes, indicating that they are complete transcripts of the rearranged foreign gene and not due to transcription of the unrearranged construct. No hybridization could be detected when a DNA fragment from the Jh-CK intron was used as probe (Fig. 1; data not shown). These results indicate that the rabbit transcripts are correctly spliced and polyadenylylated. Tail
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