HUMAN ARTIFICIAL CHROMOSOME CONTAINING HUMAN ANTIBODY LAMBDA LIGHT CHAIN GENE AND NON-HUMAN ANIMAL CONTAINING THE HUMAN ARTIFICIAL CHROMOSOME CAPABLE OF GENETIC TRANSMISSION

Abstract

The present invention relates to a human artificial chromosome which is genetically transmissible to the next generation with high efficiency and the method for using the same. More specifically, the present invention relates to: a human artificial chromosome in which an about 3.5 Mb to about 1 Mb region containing an antibody λ light chain gene derived from human chromosome 22 is bound to a chromosome fragment which is transmissible to a progeny through a germ line of a non-human animal, said chromosome fragment is derived from another human chromosome; a non-human animal carrying the human artificial chromosome and an offspring thereof; a method for producing the non-human animal; a method for producing a human antibody using the nonhuman animal or an offspring thereof; and a human antibody-producing mouse carrying the human artificial chromosome.

Claims

1. A human antibody-producing mouse, wherein an unrearranged human antibody heavy chain locus, an unrearranged human antibody κ light chain locus, and an unrearranged human antibody λ light chain locus are carried, at least both alleles of endogenous antibody heavy chain and κ light chain are disrupted or inactivated, and a human antibody heavy chain comprising a human antibody Ig γ isotype, a human antibody κ light chain, and a human antibody λ light chain are expressed in serum.

2. The human antibody-producing mouse of claim 1, which carries at least 40% of the variable region of the human antibody κ light chain.

3. The human antibody-producing mouse of claim 1, which carries all the variable regions of the human antibody heavy chain, the human antibody κ light chain, and the human antibody λ light chain.

4. The human antibody-producing mouse of claim 1, wherein a human antibody heavy chain locus, a human antibody κ light chain locus, and a human antibody λ locus are retained on a chromosome fragment derived from a human.

5. The human antibody-producing mouse of claim 1, wherein the human antibody heavy chain locus and the human antibody λ light chain locus are retained on ΔHAC or ΔΔHAC.

6. The human antibody-producing mouse of claim 1, wherein the human antibody κ light chain locus is retained on a chromosome fragment derived from a human.

7. The human antibody-producing mouse of claim 1, wherein the human antibody κ light chain locus is inserted into a mouse chromosome.

8. The human antibody-producing mouse of claim 1, which is not a chimeric mouse.

9. A human antibody producing mouse, which carries (a) an unrearranged human antibody heavy chain locus, (b) an unrearranged human antibody κ light chain gene locus and (c) and unrearranged human antibody λ light chain gene locus, wherein at least one of (a), (b) and (c) is retained on a human derived chromosome fragment(s) and at least one of (a), (b) and (c) is inserted into a mouse chromosome, and further wherein at least both alleles of endogenous heavy chains and both alleles of endogenous k light chains are inactivated, and the mouse expresses human antibody heavy chain, human antibody κ light chain and human antibody λ light chain in the serum of the mouse.

10. The human antibody producing mouse of claim 9, wherein the mouse carries an unrearranged human antibody heavy chain gene locus retained on a human derived chromosome fragment(s), an unrearranged human antibody κ light chain gene locus inserted into a mouse chromosome, and a human antibody λ light chain gene locus on a human derived chromosome fragment(s).

11. The human antibody-producing mouse of claim 9, wherein the human antibody heavy chain gene locus is retained on a human derived chromosome fragment(s).

12. The human antibody producing mouse of claim 11, wherein the human antibody heavy chain gene locus is retained on ΔHAC or ΔΔHAC.

13. The human antibody producing mouse of claim 9, wherein the human antibody λ light chain gene locus is retained on a human derived chromosome fragment(s).

14. The human antibody producing mouse of claim 13, wherein the human antibody λ light chain gene locus is retained on ΔHAC or ΔΔHAC.

15. The human antibody producing mouse of claim 9, wherein the human antibody W light chain gene locus is inserted into the mouse chromosome.

16. The human antibody producing mouse of claim 15, wherein the human antibody is transgene KCo5 is inserted into the mouse chromosome.

17. The human antibody producing mouse of claim 9, wherein the human antibody heavy chain gene locus and λ light chain gene locus are retained on ΔHAC or ΔΔHAC, and human antibody κ transgene KCo5 is inserted into the mouse chromosome.

18. The human antibody-producing mouse of claim 9, wherein the mouse is not a chimera mouse.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0070] FIG. 1 shows the production of human artificial chromosomes ΔHAC and ΔΔHAC.

[0071] FIG. 2 shows a cassette vector pTELhisD.

[0072] FIG. 3 shows a targeting vector pTELhisDλI.

[0073] FIG. 4 shows a targeting vector p553loxPHyg.

[0074] This specification includes part or all of the contents as disclosed in the specification of Japanese Patent Application No. 2001-142371, which is a priority document of the present application.

BEST MODE FOR CARRYING OUT THE INVENTION

[0075] The present invention will be described in more detail with reference to the following examples although the present invention is not limited to these examples.

[0076] The following Example 1 to Example 14 describe the production of human artificial chromosomes ΔHAC and ΔΔHAC, which are prepared by translocating and cloning 2.5 Mb and 1.5 Mb peripheral regions of the antibody λ light chain gene on human chromosome 22 to SC20 chromosome vector (FIG. 1). Further, introduction of each of the produced HACs into an individual mouse and transmission of HAC to an offspring of a chimeric mouse are described.

[Example 1] Production of Cassette Vector pTELhisD

[0077] A cassette vector pTELPuro (Kuroiwa et al., Nature Biotech., 18: 1086-, 2000) was cleaved with a restriction enzyme NotI (Boehringer) and blunt-ended using the DNA Blunting kit (Toyobo Co., Ltd.) at 72° C. for 5 minutes. After blunting, bacteria-derived alkaline phosphatase (Takara Shuzo Co., Ltd.) was used for dephosphorylation at 65° C. for 1 hour. Thereafter, a restriction enzyme BglII linker (Takara Shuzo Co., Ltd.) was added, and a ligation kit (Takara Shuzo Co., Ltd.) was used to perform ligation. Thus, plasmid pTELBg was produced in which PGKPuro cassette in pTELPuro plasmid was substituted with the BglII linker. This plasmid was cleaved with a restriction enzyme BglII and dephosphorylated in the same manner. Thereafter, it was purified by gel filtration using CHROMA SPIN-TE 400 (Clontech). Subsequently, a hisD fragment, which was cleaved out from plasmid #1-132 (distributed by Professor Shun-ichi Takeda, Kyoto University) with a restriction enzyme BamHI, was added to perform a ligation reaction in the same manner. Thus, a cassette vector pTELhisD was produced in which the PGKPuro cassette in the pTELPuro plasmid was substituted with the hisD cassette (FIG. 2).

[Example 2] Production of Targeting Vector pTELhisDλI

[0078] A targeting vector pTELhisDλI for inserting a human telomeric sequence into the AP000344 region located very close to the Ig λ locus on human chromosome 22 and on the telomeric side (about 400 Kb telomeric side) was produced in the following manner. At the outset, the AP000344 genomic region was amplified by PCR using the primers below.

TABLE-US-00001 1269D1-F;  (SEQ ID NO: 1) 5′-TCGAGGATCCGACAAGTTCTCTTCTCTTTTCCTTCTGCCC-3′ 1269D1-R;  (SEQ ID NO: 2) 5′-TCGAGGATCCGCTGCTAAGCTACTGTTCTCTTTTTTCCCC-3′

[0079] PCR was carried out using GeneAmp 9600 (manufactured by Perkin-Elmer) as a thermal cycler and LA Taq (Takara Shuzo Co., Ltd.) as the Taq polymerase, and the attached buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used in accordance with the recommended conditions. Regarding the temperature and cycle conditions, after thermal denaturation at 94° C. for 1 minute, 35 cycles of 98° C. for 10 seconds and 68° C. for 11 minutes were carried out. The PCR product was treated with protenase K (Gibco) and then subjected to gel filtration with CHROMA SPIN-TE 400 (Clontech). Thereafter, the PCR product was cleaved with a restriction enzyme BamHI (Boehringer) and then subjected to gel filtration with CHROMA SPIN-TE 1000 (Clontech). This PCR fragment was cloned into the BamHI site of the plasmid pTELhisD. Since the direction of the AP000344 genomic sequence was from centromere to telomere, the cloned AP000344 genomic fragment in the same direction as the human telomere sequence was determined as a targeting vector pTELhisDλI of interest (FIG. 3).

[Example 3] Production of Targeting Vector p553 loxPHyg

[0080] A targeting vector p553loxPHyg for inserting loxP sequence which is a recognition sequence of a Cre recombinant enzyme into the AP000553 region located very close to the Ig λ locus on human chromosome 22 and on the centromeric side (about 300 Kb centromeric side) was produced in the following manner. At the outset, the AP000553 genomic region was amplified by PCR using the primers below.

TABLE-US-00002 553-F3;  (SEQ ID NO: 3) 5′-TCGAGTCGACTGTAGCTGACTTTAGCCACCCACAAGTAC-3′ 553-R3; (SEQ ID NO: 4) 5′-TCGAGTCGACCTTGCTGATTATACCTCATCTCCTTCCCTC-3′

[0081] PCR was carried out using GeneAmp 9600 (manufactured by Perkin-Elmer) as a thermal cycler and LA Taq (Takara Shuzo Co., Ltd.) as the Taq polymerase, and the attached buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used in accordance with the recommended conditions. Regarding the temperature and cycle conditions, after thermal denaturation at 94° C. for 1 minute, 35 cycles of 98° C. for 10 seconds and 68° C. for 15 minutes were carried out. The PCR product was treated with protenase K (Gibco) and then subjected to gel filtration with CHROMA SPIN-TE 400 (Clontech). Thereafter, the PCR product was cleaved with a restriction enzyme SalI (Boehringer) and then subjected to gel filtration with CHROMA SPIN-TE 1000 (Clontech). This PCR fragment was cloned into the SalI site of plasmid pBluescriptII (the NotI site was previously deleted and then the SrfI linker was inserted into the SacII site) (pBS553). Subsequently, pBS553 was cleaved with a restriction enzyme HpaI (Boehringer) and dephosphorylated, and the NotI linker was then inserted by ligation (pBS553N). After pBS553N was cleaved with a restriction enzyme NotI and dephosphorylated, a DNA fragment containing loxP was cleaved out with a restriction enzyme NotI (Boehringer) from a cassette vector ploxPHyg, followed by ligation. A vector having the loxP sequence in the same direction as the cloned AP000553 genomic fragment was determined as a targeting vector p553loX:PHyg (FIG. 4).

[Example 4] Site-Specific Cleavage of Human Chromosome 22 in Chicken DT-40 Cell

[0082] The targeting vector pTELhisDλI produced in Example 2 was transfected into the chicken DT-40 cell (clone 52-18) retaining a full length of human chromosome 22 produced by the method described in WO 98/37757 and the DT-40 cell (clone HF38) retaining a fragment of human chromosome 22 that was already cleaved at the LIF locus, and the human telomeric sequence was inserted into the AP000344 genomic region to attempt the cleavage of chromosome 22 at the insertion site.

[0083] The chicken DT-40 cell was cultured in RPMI 1640 medium (Gibco) comprising 10% fetal bovine serum (Gibco, hereinafter referred to as “FBS”), 1% chicken serum (Gibco), and 10.sup.−4M 2-mercaptoethanol (Sigma) added therein. About 10.sup.7 cells were washed once with additive-free RPMI 1640 medium and suspended in 0.5 ml of additive-free RPMI 1640 medium. 25 to 30 μg of targeting vector pTELhisDλI, which has been linearized with a restriction enzyme SrfI (Toyobo Co., Ltd.), was added, transferred into a cuvette (Bio-Rad) for electroporation, and allowed to stand at room temperature for 10 minutes. The cuvette was set in a Gene Pulser (Bio-Rad), and voltage was applied at 550 V, 25 μF. After the cuvette was allowed to stand at room temperature for 10 minutes, it was cultured for 24 hours. Twenty four hours later, the medium was exchanged with a medium containing histidinol (0.5 mg/ml), the culture solution was fractionated to ten 96-well culture plates, and selective culture was carried out for about 2 weeks. Genomic DNA was extracted from a histidinol-resistant clone using the Puregene DNA Isolation Kit (CentraSystem), and cleavage of human chromosome 22 in the AP000344 genomic region was confirmed by PCR using primers for detecting HCF2 (Kuroiwa et al., Nature Biotech. 18: 1086, 2000), Igλ (Tomizuka et al., Nature Genet., 16: 133, 1997), D22S1174, D22S315, D22S275 (BIOS), and LIF (Kuroiwa et al., Nucleic Acid Research, 26: 3447-3448, 1998).

[0084] PCR was carried out using GeneAmp 9600 (manufactured by Perkin-Elmer) as a thermal cycler and LA Taq (Takara Shuzo Co., Ltd.) as the Taq polymerase, and the attached buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used in accordance with the recommended conditions. Regarding the temperature and cycle conditions, after thermal denaturation at 94° C. for 1 minute, 35 cycles of 98° C. for 10 seconds, 56° C. for 30 seconds, and 72° C. for 30 seconds were carried out. When clone 52-18 was transfected, 48 clones were screened and 1 clone (T32) was found to be a clone of interest. When HF38 was transfected, 96 clones were screened and 2 clones (HT69, HT72) were found to be clones of interest.

[0085] Further, whether chromosome 22 was cleaved in the AP000344 region or not was confirmed by FISH analysis.

[0086] In order to visually judge that human chromosome 22 was cleaved in the AP000344 genomic region, FISH analysis was carried out using a probe capable of detecting a hisD resistant gene in a targeting vector. The method was in accordance with Kuroiwa et al. (Nucleic Acid Research, 26: 3447-3448, 1998). Based on COT1 staining (rhodamine label, red), chromosome 22 was found to be fragmented in T32, HT69, and 1HT72 compared to a full-length human chromosome 22. Further, a signal (FITC label, yellow) derived from a hisD probe was detected at the telomeric end. This indicates that AP000344 into which a targeting vector had been inserted is the telomeric end of a fragment of chromosome 22.

[0087] From the above result, it was concluded for T32, HT69, and HT72 that human chromosome 22 was cleaved in the AP000344 region.

[Example 5] Site-Specific Insertion of loxPHyg Cassette on Human Chromosome 22 in Chicken DT-40 Cell

[0088] In the above HT69 and 72, the loxP sequence is already inserted into the HCF2 locus (about 1 Mb centromeric side from the Ig λ locus). Therefore, in clone T32, the targeting vector p553loxPHyg produced in Example 3 was transfected into the AP000553 region located very close to the Ig λ locus and on the centromeric side (about 300 Kb centromeric side) to attempt the insertion of the loxP sequence.

[0089] In the same manner as described above, a targeting vector p553loxPHyg, which has been linearized with a restriction enzyme SrtI (Toyobo Co., Ltd.), was transfected into clone T32 and selective culture was conducted in a medium containing hygromycin B (1 mg/ml) for about 2 weeks. Genomic DNA was extracted from a hygromycin B-resistant clone and a homologous recombinant was identified by PCR using the 2 sets of primers below.

TABLE-US-00003 (SEQ ID NO: 5) 553-F4; 5′-GCTAAGGCACTTCGGTTCTCTTTGTGTTC-3′ (SEQ ID NO: 6) 553-R4; 5′-GGTTGTCTTTAAAAGCAGGGATAAGGATG-3′ (SEQ ID NO: 7) 553-F5; 5′-AGAAGAAAGGAGTGGGTGCTAAACATTCAG-3′ (SEQ ID NO: 8) 553-R5; 5′-GGTTAGATGGCACCAAATGAAAGGAGAAG-3′

[0090] PCR was carried out using GeneAmp 9600 (manufactured by Perkin-Elmer) as a thermal cycler and LA Taq (Takara Shuzo Co., Ltd.) as the Taq polymerase, and the attached buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used in accordance with the recommended conditions. Regarding the temperature and cycle conditions, after thermal denaturation at 94° C. for 1 minute, 35 cycles of 98° C. for 10 seconds and 68° C. for 15 minutes were carried out. As a result of screening of 69 clones, 3 clones (553-2, 6, 14) were identified as homologous recombinants.

[Example 6] Construction of Human Artificial Chromosome ΔHAC Prepared by Translocating and Cloning 2.5 Mb Periphery of Human Antibody λ Light Chain Gene Region (HCF2-Ig λ-AP000344) to SC20 Chromosome Vector

[0091] At the outset, clone HT72 obtained in Example 4 was subjected to cell fusion with clone R of the DT-40 cell retaining the SC20 chromosome vector (Kuroiwa et al., Nature Biotech. 18: 1086, 2000) to produce a DT-40 hybrid retaining both a fragment of human chromosome 22 and an a fragment of chromosome 14 (SC20 chromosome vector). [0092] (1) Production of DT-40 Hybrid Retaining Both a Fragment of Human Chromosome 22 and SC20 Chromosome Vector

[0093] Clone R was cultured in RPMI 1640 medium containing blasticidin S (10 μg/ml) and clone HT72 was cultured in RPMI 1640 medium containing hygromycin B (1 mg/ml). Both clones were mixed with each other in amounts of 1 to 2×10.sup.7 respectively and centrifuged, and then washed twice with a serum-free RPMI 1640 medium. After the residual medium was completely removed, 0.5 ml of 50% PEG 1500 (Boehringer), which was preheated at 37° C., was gently added, and the mixture was vigorously mixed using a pipette for about 2 minutes. Thereafter, 1 ml of serum-free RPMI 1640 medium was slowly added over a period of 1 minute, 9 ml of serum-free RPMI 1640 medium was then added over a period of about 3 minutes; and the mixture was allowed to stand at 37° C. for 10 minutes. Thereafter, the mixture was centrifuged at 1,200 rpm for 5 minutes and cultured for 24 to 48 hours in a serum-containing RPMI 1640 medium. Thereafter, the medium was exchanged with RPMI 1640 medium containing blasticidin S (10 μg/ml) and hygromycin B (1 mg/ml); and the culture solution was fractionated to five 24-well culture plates, followed by culturing for 3 to 4 weeks. Genomic DNA was extracted from the double-resistant clone, and PCR was carried out using the primers below to confirm that two fragments, i.e., a fragment of human chromosome 14 (SC20 chromosome vector) and a fragment of chromosome 22, were retained.

Primers for Detecting Human Chromosome 14

[0094]

TABLE-US-00004 (SEQ ID NO: 9) VH3-F; 5′-AGTGAGATAAGCAGTGGATG-3′ (SEQ ID NO: 10) VH3-R; 5′-GTTGTGCTACTCCCATCACT-3′

Primers for Detecting Human Chromosome 22

[0095]

TABLE-US-00005 (SEQ ID NO: 11) Igλ-F; 5′-GAGAGTTGCAGAAGGGGTGACT-3′ (SEQ ID NO: 12) Igλ-R; 5′-GGAGACCACCAAACCCTCCAAA-3′

[0096] PCR was carried out using GeneAmp 9600 (manufactured by Perkin-Elmer) as a thermal cycler and Ex Taq (Takara Shuzo Co., Ltd.) as the Taq polymerase, and the attached buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used in accordance with the recommended conditions. Regarding the temperature- and cycle conditions, after thermal denaturation at 94° C. for 1 minute, 35 cycles of 98° C. for 10 seconds, 56° C. for 30 seconds, and 72° C. for 30 seconds were carried out. As a result of PCR, 6 clones (56HT2, 3, 4, 5, 6, 7) were found positive. Further, the result of FISH analysis using human COTl DNA as a probe demonstrated that all these clones retained two fragments of human chromosome independently from each other. Based on the above results, these 6 hybrid clones were judged to retain two fragments, i.e., a fragment of human chromosome 14 (SC20 chromosome vector) and a fragment of chromosome 22.

[0097] (2) Site-Specific Translocation of 2.5 Mb Region of Human Chromosome 22 (HCF2-Igλ-AP000344) to SC20 Chromosome Vector in DT-40 Hybrid Clone (56HT2)

[0098] (2)-1 Construction of Stable Expression Vector for Cre Recombinant Enzyme

[0099] In accordance with the method by Kuroiwa et al. (described above), site-specific translocation between human chromosomes was carried out using the Cre-loxP system. Since recombination efficiency between non-homologous chromosomes was expected to be very low even in this system, it was considered that the Cre enzyme should be stably expressed instead of being transiently expressed. Thus, the following type of expression vector was constructed.

[0100] A PGKPuro fragment which was cleaved out by EcoRI from a plasmid of which the NotI site in plasmid PGKPuro (distributed by Dr. Peter W. Laird, WHITEHEAD INSTITUTE) had been substituted by the EcoRI site, was cloned into the EcoRI site in a Cre recombinase expression vector: pBS185 (Gibco) (pBS185Puro).

[0101] (2)-2 Site-Specific Translocation of 2.5 Mb Region of Human Chromosome 22 (HCF2-Igλ-AP000344) to SC40 Chromosome Vector in DT-40 Hybrid Clone Using Cre-loxP System

[0102] In the same manner as described above, a stable Cre recombinant enzyme expression vector: pBS185Puro which had been linearized with a restriction enzyme KpnI (Boehringer) was transfected into the 56HT2 hybrid clone, the culture solution was fractionated to a 24-well plate, and selective culture was conducted in the presence of puromycin (3 μg/ml) for about 2 weeks. Genomes were extracted from each well, and nested PCR using the two sets of primers below was carried out to determine whether or not translocation between the SC20 chromosome vector and a fragment of human chromosome 22 had occurred.

TABLE-US-00006 (SEQ ID NO: 13) PGK-1; 5′-ATAGCAGCTTTGCTCCTTCG-3′ (SEQ ID NO: 14) GFP-1; 5′-TTCTCTCCTGCACATAGCCC-3′ (SEQ ID NO: 15) PGK-2; 5′-TGTTCTCCTCTTCCTACTCTCC-3′ (SEQ ID NO: 16) GFP-2; 5′-TGAAGGTAGTGACCAGTGTTGG-3′

[0103] PCR was carried out using GeneAmp 9600 (manufactured by Perkin-Elmer) as a thermal cycler and Ex Taq (Takara Shuzo Co., Ltd.) as the Taq polymerase, and the attached buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used in accordance with the recommended conditions. As the first PCR, after thermal denaturation at 94° C. for 1 minute, 35 cycles of 98° C. for 10 seconds, 61° C. for 30 seconds, and 72° C. for 1 minute were carried out using PGK-1 and GFP-1 as primers. Using a part of this reaction solution as a template, 35 cycles of 98° C. for 10 seconds, 59° C. for 30 seconds, and 72° C. for 30 seconds were then carried out using PGK-2 and GFP-2 as primers. A cell pool in a well which found by PCR to be translocated was cultured until the cell number reached 10.sup.7, and the pool was suspended in 4 ml of PBS (phosphate buffered saline-solution) having 5% FBS and 1 μg/ml propidium iodide (PI) added therein and analyzed by FACS Vantage (Becton Dickinson). As reported by Kuroiwa et al. (described above), when recombination or translocation between loxPs occurred, the GFP gene is reconstructed and expressed. Thus, translocated cells can be detected by FACS. Sorting of cell fractions which were considered to be GFP positive was repeated twice. Culture after every sorting operation was performed in RPMI 1640 medium containing hygromycin B (1 mg/ml). As a result, GFP positive cells were concentrated at a purity of 98 to 99%.

[0104] Subsequently, whether or not recombination between loxPs in GFP positive clone (ΔH21), which was cloned by FACS, had occurred as expected was confirmed by PCR using PGK-2 and GFP-2 as primers. Further, clone ΔH21 was subjected to FISH analysis (Kuroiwa et al., described above) using a human chromosome 14-specific probe (rhodamine label) and a human chromosome 22-specific probe (FITC label). As a result, the existence of an artificial chromosome in which the human chromosome 22 region was clearly translocated to the SC20 chromosome vector (a fragment of human chromosome 14) was confirmed.

[0105] Based on the above result, it was concluded for clone ΔH21 that a human artificial chromosome ΔHAC was constructed in which 2.5 Mb of periphery of human antibody λ light chain gene region (HCF2-Igλ-AP000344) was translocated and cloned to the SC20 chromosome vector.

[0106] A chicken DT-40 cell (ΔHAC) retaining ΔHAC was deposited internationally at the International Patent Organism. Depositary of the National Institute of Advanced Industrial Science and Technology (Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan) as of May 9, 2001, under the accession number of FERM-BP-7582.

[Example 7] Construction of Human Artificial Chromosome ΔΔHAC Prepared by Translocating and Cloning 1.5 Mb Periphery of Human Antibody λ Light Chain Gene Region (AP000553-Igλ-AP000344) to SC20 Chromosome Vector

[0107] In the above ΔHAC, about 1 Mb of extra region still remains between Ig λ and HCF2. For the purpose of strictly removing an extra chromosome region and translocating and cloning only the periphery of an Ig λ gene region, it was attempted to construct human artificial chromosome ΔHAC in which 1.5 Mb of AP000553-Igλ-AP000344 region was translocated and cloned to the SC20 chromosome vector.

[0108] At the outset, clone 553-2 obtained in Example 5 was subjected to cell fusion with the clone R to produce the DT-40 hybrid retaining both the fragment of human chromosome 22 and chromosome 14 (SC20 chromosome vector).

[0109] (1) Production of DT-40 Hybrid Retaining Both the Fragment of Human Chromosome 22 and SC20 Chromosome Vector

[0110] The clone R was cultured in RPMI 1640 medium containing blasticidin S (10 μg/ml) and the clone 553-2 was cultured in RPMI 1640 medium containing hygromycin B (1 mg/ml). Both clones were mixed with each other in amounts of 1 to 2×10.sup.7, respectively and centrifuged, and then washed twice with a serum-free RPMI 1640 medium. After the residual medium was completely removed, 0.5 ml of 50% PEG 1500 (Boehringer), which was preheated at 37° C., was gently added, and the mixture was vigorously mixed using a pipette for about 2 minutes. Thereafter, 1 ml of serum-free RPMI 1640 medium was slowly added over a period of 1 minute, 9 ml of serum-free RPMI 1640 medium was then added over a period of about 3 minutes, and the mixture was allowed to stand at 37° C. for 10 minutes. Thereafter, the mixture was centrifuged at 1,200 rpm for 5 minutes and cultured for 24 to 48 hours in a serum-containing RPMI 1640 medium. Thereafter, the medium was exchanged with RPMI 1640 medium containing blasticidin S (10 μg/ml) and hygromycin B (1 mg/ml), and the culture solution was fractionated to five 24-well culture plates, followed by culturing for 3 to 4 weeks. Genomic DNA was extracted from an obtained hybrid clone (for example, clone 553R1), and PCR was carried out using the same primers as used in Example 6 to confirm that two fragments, i.e., the fragment of human chromosome 14 and the fragment of chromosome 22, were retained. Further, FISH analysis was carried out using human COT1 DNA as a probe and two fragments of human chromosome were confirmed to exist independently from each other. Based on the above experiment, it was concluded that hybrid clone 553R1 retained two fragments, i.e., the fragment of human chromosome 14 (SC20 chromosome vector) and the fragment of chromosome 22.

[0111] (2) Site-Specific Translocation of 1.5 Mb Region of Human Chromosome 22 (AP000553-Igλ-AP000344) to SC20 Chromosome Vector in DT-40 Hybrid Clone (553R1)

[0112] In the same manner as described above, a stable Cre recombinant enzyme expression vector: pBS18SPuro which had been linearized with a restriction enzyme κpnI (Boehringer) was transfected into the hybrid clone 553R1, the culture solution was fractionated to a 12-well plate, and selective culture was conducted in the presence of puromycin (3 μg/ml) for about 2 weeks. Genomes were extracted from each well, and nested PCR using the two sets of primers below was carried out to determine whether or not translocation between the SC20 chromosome vector and a fragment of human chromosome 22 had occurred.

TABLE-US-00007 (SEQ ID NO: 13) PGK-1; 5′-ATAGCAGCTTTGCTCCTTCG-3′ (SEQ ID NO: 14) GFP-1; 5′-TTCTCTCCTGCACATAGCCC-3′ (SEQ ED NO: 15) PGK-2; 5′-TGTTCTCCTCTTCCTACTCTCC-3′ (SEQ ID NO: 16) GFP-2; 5′-TGAAGGTAGTGACCAGTGTTGG-3′

[0113] PCR was carried out using GeneAmp 9600 (manufactured by Perkin-Elmer) as a thermal cycler and Ex Taq (Takara Shuzo Co., Ltd.) as the Taq polymerase, and the attached buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used in accordance with the recommended conditions. As the first PCR, after thermal denaturation at 94° C. for 1 minute, 35 cycles of 98° C. for 10 seconds, 61° C. for 30 seconds, and 72° C. for 1 minute were carried out using PGK-1 and GFP-1 as primers. Using a part of this reaction solution as a template, 35 cycles of 98° C. for 10 seconds, 59° C. for 30 seconds, and 72° C. for 30 seconds were carried out using PGK-2 and GFP-2 as primers. Cell pools (2 pools: DDH5, 6) in a well which were found by PCR to be translocated increased until the cell number reached 10.sup.7, and the pool was suspended in 4 ml of PBS (phosphate buffered saline solution) having 5% FBS and 1 μg/ml propidium iodide (PI) added therein and analyzed by FACS Vantage (Becton Dickinson). Sorting of cell fractions which were considered to be GFP positive was repeated twice. Culture after every sorting operation was performed in RPMI 1640 medium containing hygromycin B (1 mg/ml). As a result, GFP positive cells were concentrated at a purity of 98 to 99%.

[0114] Subsequently, whether or not recombination between loxPs in GFP positive clones (ΔΔHS, 6), which were cloned by FACS, had occurred as expected was confirmed by PCR using PGK-2 and GFP-2 as primers. Further, clones ΔΔH5, 6 were subjected to FISH analysis (Kuroiwa et al., described above) using a human chromosome 14-specific probe (rhodamine label) and a human chromosome 22-specific probe (FITC label). As a result, the existence of an artificial chromosome in which a region of human chromosome 22 was clearly translocated to the SC20 chromosome vector (a fragment of human chromosome 14) was confirmed for both clones.

[0115] Based on the above result, it was concluded for the two clones ΔΔH5, 6 that human artificial chromosome ΔΔHAC was constructed in which 1.5 Mb periphery of human antibody λ light chain gene region (AP000553-Igλ-AP000344) was translocated and cloned to the SC20 chromosome vector.

[0116] A chicken DT-40 cell (ΔΔHAC) retaining ΔΔHAC was deposited internationally at the International Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology (Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan) as of May 9, 2001, under the accession number of FERM-BP-7581.

[Example 8] Cell Fusion Between ΔHAC-Containing DT-40 Hybrid Cell and Chinese Hamster CHO Cell

[0117] As reported by Kuroiwa et al. (described above), introduction of a constructed HAC into a CHO cell was first attempted to introduce a constructed HAC into a mouse ES cell. However, the ΔHAC-containing DT-40 hybrid cell ΔH21 had a low microcell-forming ability and, thus, introduction of ΔHAC into the CHO cell by a microcell method was not successful (WO 00/10383). Thus, introduction of ΔHAC into the CHO cell through cell fusion between the ΔHAC-containing DT-40-hybrid cell ΔH21 and the CHO cell was newly attempted.

[0118] 1 to 2×10.sup.7 ΔH21 clones and 1×10.sup.7 CHO cells were mixed and centrifuged, and the mixture was then washed twice with a serum-free DMEM medium. After the residual medium was completely removed, 0.5 ml of 50% PEG 1500 (Boehringer), which was preheated at 37° C., was gently added, and the mixture was vigorously mixed using a pipette for about 2 minutes. Thereafter, 1 ml of serum-free DMEM medium was slowly added over a period of 1 minute, 9 ml of serum-free DMEM medium was then added over a period of about 3 minutes, and the mixture was allowed to stand at 37° C. for 10 minutes. Thereafter, the mixture was centrifuged at 1,200 rpm for 5 minutes and cultured in a serum-containing FI2 medium (Gibco) for 24 hours. Thereafter, the medium was exchanged with a f12 medium containing G418 (1 mg/ml) and hygromycin B (0.6 mg/ml), and the culture solution was fractionated to three 24-well culture plates, followed by culturing for 3 to 4 weeks.

[0119] Genomes were extracted from the resistant clone, and PCR was carried out using primers for detecting VH3 and Ig λ in the same manner as in Example 6. As a result, 2 clones (DI5, ΔC30) were found PCR positive. Further, these 2 clones were subjected to FISH analysis by double staining using a human chromosome 14-specific probe and a human chromosome 22-specific probe in the same manner as in Example 6 to confirm the existence of ΔHAC. Regarding cell fusion between DT40 and CHO, most of chromosomes derived from DT40 were dropped out and the karyotype was substantially the same as that of the wild-type CHO cell. This eventually enabled the production of the CHO clone retaining ΔHAC through cell fusion between the DT40 cell and the CHO cell. This indicated the possibility that the cell fusion might be useful as an alternative method for the case where the microcell-forming ability of DT40 clone was low.

[Example 9] Introduction of ΔΔHAC from ΔΔHAC-Containing DT-40 Hybrid Cell into CHO Cell

[0120] The microcell-forming ability of ΔΔHAC-containing DT-40 hybrid clones ΔΔH5, 6 was not insufficient and, thus, the microcell method was employed as reported by Kuroiwa et al. (described above).

[0121] DT-40 hybrid clones ΔΔH5, 6 were respectively cultured in eight T225 flasks (Sumiron), and the medium was exchanged with RPMI 1640 medium having 20% FBS, 1% chicken serum, 10.sup.−4 M 2-mercaptoethanol, and 0.05 μg/ml colcemid added therein when the content of the flasks became confluent. Cells were cultured for an additional 24 hours to form microcells. The cells were suspended in 24 ml of serum RPMI 1640 medium, fractionated in an amount of 2 ml each to twelve 25 cm.sup.2 flasks for centrifugation (Corning) which were precoated with 100 μg/ml poly L-lysin, and cultured at 37° C. for 1 hour. The cells were then allowed to adhere on the bottoms of the flasks. The culture solution was removed, and a solution of cytochalasin B (10 μg/ml, Sigma), which was preheated at 37° C., was filled into a flask for centrifugation and subjected to centrifugation at 34° C. at 8,000 rpm for 1 hour. Microcells were suspended in a serum-free DMEM medium and purified through 8 μm, 5 μm, and 3 μm filters. After purification, the microcells were centrifuged at 1,700 rpm for 10 minutes and suspended in 5 ml of serum-free DMEM medium. Separately, about 10′ CHO cells were peeled by trypsin treatment, washed twice with serum-free DMEM medium, and suspended in 5 ml of serum-free DMEM medium. The microcells were recentrifuged at 1,700 rpm for 10 minutes, and 5 ml of the above CHO suspension was gently superposed thereon without removing the supernatant. After centrifugation, the culture solution was removed, 0.5 ml of PEG 1500 solution (Boehringer) was added, and the mixture was vigorously stirred using a pipette for about 2 minutes. Thereafter, 10 ml of serum-free DMEM medium was slowly added over a period of about 3 minutes and the mixture was allowed to stand at 37° C. for 10 minutes. After centrifugation, cells were suspended in F12 medium containing 10%. FBS (Gibco) and fractionated to five to six 24-well culture plates, followed by culturing at 37° C. for 24 hours. Thereafter, the medium was exchanged with the F12 medium containing 800 μg/ml G418 and selective culture was conducted for 3 to 4 weeks.

[0122] Genomic DNA was extracted from the G418-resistant clone, and PCR was carried out using primers for detecting Ig λ and VH3 and PGK-2 and GFP-2 primers under the same conditions as described above to identify a CHO clone retaining ΔΔHAC (for example, ΔΔC10, 13). Further, the clones which were found positive by PCR were subjected to FISH analysis using a human chromosome 14-specific probe and a human chromosome 22-specific probe to visually confirm the existence of ΔΔHAC. Based on these results, it was concluded that the clones of CHO cell retaining ΔΔHAC were obtained.

[Example 10] Introduction of ΔHAC or ΔΔHAC from CHO Cell into Mouse ES Cell

[0123] In order to produce a chimeric mouse carrying ΔHAC or ΔΔHAC, ΔHAC or ΔΔHAC was transduced from the CHO cell retaining ΔHAC or ΔΔHAC obtained in Examples 8 or 9 to the mouse ES cell (wild-type TT2F) by the microcell method.

[0124] In accordance with the method by Tomizuka et al. (Nature Genet. 16: 133, 1997), microcells were purified from the CHO cells (D15, ΔΔC10, ΔΔC13 or the like) retaining about 10.sup.8 ΔHAC or ΔΔHAC and suspended in 5 ml of DMEM. About 10 mouse ES cells TT2F were peeled by trypsin treatment, washed three times with DMEM, suspended in 5 ml of DMEM, added to the centrifuged microcells, and centrifuged at 1,250 rpm for 10 minutes. The supernatant was then completely removed. The precipitate was thoroughly loosened by tapping, 0.5 ml of 1:1.4 PEG solution [a solution of 5 g PEG 1000 (Wako Pure Chemicals Industries Ltd.) and 1 ml of DMSO (Sigma) in 6 ml of DMEM] was added, and the mixture was thoroughly stirred for about 1 minute and 30 seconds. Thereafter, 10 ml of DMEM was slowly added, the mixture was centrifuged at 1,250 rpm for 10 minutes and suspended in 30 ml of ES medium and fractionated to three petri dishes (Corning, diameter 100 mm) previously loaded with feeder cells, followed by culturing. The medium was exchanged with a medium containing 300 μg/ml G418 twenty-four hours later, and selective culture was conducted for about 1 week.

[0125] As a result, 14 clones were found positive from among D 15 clones (retaining ΔHAC), 8 clones from among ΔΔC10 (retaining ΔΔHAC), and 8 clones from among ΔΔC13 (retaining ΔΔHAC) by PCR using primers for detecting Ig λ and VH3. Further, as a result of FISH analysis using human COT1 DNA probe (Tomizuka et al., Nature Genet. 16: 133, 1997), the existence of ΔHAC or ΔΔHAC specifically detected by the COT1 probe was confirmed.

[0126] Based on the above results, it was concluded that 14 clones were obtained from ΔHAC-retaining TT2F cells and 16 clones from ΔΔHAC-retaining TT2F cells.

[Example 11] Production of Chimeric Mouse Carrying Human Artificial Chromosomes ΔHAC and ΔΔHAC

[0127] A chimeric mouse was produced in accordance with the method by Tomizuka et al. (Nature Genet., 16: 33, 1997) using clones of the ES cell obtained in Example 10. As a host, MCH(ICR)(white, purchased from CLEA Japan, Inc.) or a 8-cell stage embryo obtained by female-male crossing of antibody heavy chain knock-out mice (Tomizuka et al., Proc. Natl. Acad. Sci. USA, vol. 97, 722-727, 2000) was used. Whether an offspring mouse obtained by transplanting the injected embryo into the foster parent is chimeric or not can be determined based on its coat color. Wild-type TT2F/ΔHAC clones (TΔ#6, obtained in Example 10) were injected into 400 embryos and the injected embryos were transplanted into foster parents. As a result, 7 chimeric mice (a dark brown portion is recognized in coat color) were borne. More specifically, it was shown that the ES cell strain (TT2F) retaining human artificial chromosome ΔHAC has a chimera-forming ability, that is, has an ability to differentiate into normal tissues of an individual mouse.

[0128] In the same manner as described above, wild-type TT2F/ΔΔHAC clones (TΔΔ#21) obtained in Example 10 were injected into 180 embryos and the injected embryos were transplanted into the foster parents. As a result, 2 chimeric mice (a dark brown portion is recognized in coat color) were borne. One of them was an individual with a chimerism of about 100%, i.e., a white portion could not be substantially observed. More specifically, it was shown that the ES cell strain (TT2F) retaining human artificial chromosome ΔΔHAC has a chimera-forming ability, that is, has an ability to differentiate into normal tissues of an individual mouse.

[Example 12] Retention of Artificial Chromosome in Somatic Cell of Chimeric Mouse Produced from ES Cell Retaining Human Artificial Chromosomes ΔHAC and ΔΔHAC

[0129] Genomic DNA was prepared from a tail of the chimeric mouse produced in Example 11 from TT2F/ΔHAC clone (TΔ#6) (chimerism of about 85%) by the method reported by Tomizuka et al. (Nature Genet., 16: 133, 1997), and PCR was carried out using primers for detecting Ig λ and VH3 in the same manner as described “above to examine ΔHAC retention. As a result, it was found to be positive for both of the primers and ΔHAC retention in the somatic cells of the chimeric mouse was confirmed. Serum was collected from a chimeic mouse (a chimerism of about 85%) and another chimeric mouse produced from TΔ#6 (a chimerism of about 90%). and the expression of human λ chain and human λ chain proteins was examined by ELISA (Tomizuka et al., Nature Genet., 16: 133, 1997, Proc. Natl. Acad. Sci, USA, vol. 97, 722-727, 2000). As a result, human μ chain and λ chain were both positive for both of the chimeric mice.

[0130] Similarly, DNA, derived from a tail of the chimeric mouse (a chimerism of about 100%, Example 11) derived from the ES cell clone (TΔΔ#21) retaining ΔΔHAC, was found positive for the above two primers and ΔΔHAC retention was confirmed. Further, ELISA analysis similar to the above indicates that both human μ chain and λ chain are positive in serum of the ΔΔHAC-carrying chimeric mouse.

[0131] The chimerism of the λHAC carrying chimeric mouse obtained from the ES cell retaining λHAC was about 80% at the maximum, however, chimeric mice with chimerisms of about 85% and 90% were obtained from ΔHAC and a chimeric mouse with a chimerism of about 100% was obtained from ΔΔHAC. Use of a chimeric mouse with a higher chimerism can result in differentiation of introduced chromosome retaining ES cell into germ cells with higher efficiency and genetic transmission of the introduced chromosome. That is, use of ΔHAC and ΔΔHAC can be expected to enhance the genetic transmission efficiency of a fragment of human chromosome 22 containing an antibody immunoglobulin λ chain gene in a mouse.

[Example 13] Genetic Transmission of Artificial Chromosome from Chimeric Mouse Carrying Human Artificial Chromosome ΔHAC and ΔΔHAC

[0132] A chimeric female mouse (chimerism of about 85%) produced in Example 11 from TT2F/ΔHAC clone (TA#6) was crossed with a male mouse MCH(ICR) (white, purchased from CLEA Japan, Inc.). Among 10 offspring mice born from the chimeric mouse, 4 had a coat color of dark brown, which indicates the retention of a dominant genotype derived from the ES cell. That is, the ES cell strain and TA#6 retaining ΔHAC were found to be differentiated into functional egg cells in a chimeric female mouse. A part of the tails of the four dark brown offspring mice was cut out and genomic DNA was prepared from the sample. The obtained DNA was subjected to PCR using primers for detecting Ig λ and VH3 in the same manner as described above. As a result of the examination on ΔHAC retention, all of the four mice were found positive for both of the primers and ΔHAC retention in the offspring of the chimeric mouse was confirmed. Further, serum was collected from 3 out of the 4 mice and expression of human ρ chain and human λ chain was examined by ELISA (Tomizuka et al., Nature Genet., 16: 133, 1997, Proc. Natl. Acad. Sci. USA., vol. 97, 722-727, 2000). As a result, all the examined three mice were found positive for both human μ chain and λ chain. Genetic transmission of ΔΔHAC from the chimeric mouse produced from clone TT2F/ΔHAC in Example 11 is indicated in the same manner.

[0133] In mouse lineages that respectively carry and genetically transmit either ΔHAC or ΔΔHAC, stable retention of each HAC is examined by FISH analysis and the like of fibroblasts prepared from tails. As a result, stable retention of each HAC in the somatic cell of the mouse lineage is shown.

[0134] In mouse lineages that carry and genetically transmit either ΔHAC or ΔΔHAC, expression of complete human antibody molecules consisting of human Ig λ chain/heavy chain is confirmed by ELISA and the like. Further, a mouse lineage that respectively carries and genetically transmits either ΔHAC or ΔΔHAC is repetitively crossed with a mouse lineage having deleted endogenous antibody heavy chain and light chain κ gene, thereby obtaining mouse lineages carrying each HAC and being homogeneous in terms of the endogenous antibody heavy chain and κ chain genes deficiency. These mouse lineages mainly produce a complete human antibody comprising the human Ig heavy chain and λ chain.

[Example 14] Construction of Mouse Lineage Simultaneously Expressing Human Immunoglobulin Heavy Chain, Light Chain λ, and Light Chain κ

[0135] A mouse lineage which simultaneously produces the human Ig heavy chain, κ light chain, and λ light chain and produces an antibody mainly composed of a molecule comprising the human Ig heavy chain and κ light chain or λ light chain can be produced by crossing between the lineage (A) and lineage (B) below.

[0136] (A) TC (ΔHAC), a mouse lineage which carries and genetically transmits ΔHAC, or TC (ΔΔHAC), a mouse lineage which carries and genetically transmits ΔΔHAC (see Example 13).

[0137] (B) TC(W23)/ΔH/Δκ, a mouse lineage which is a homozygote for the endogenous antibody heavy chain and κ chain genes deficiency and carries and genetically transmits fragment W23 of chromosome 2 (Tomizuka et al., Proc. Natl. Acad. Sci. USA., vol. 97, 722-727, 2000).

[0138] The offspring mice obtained by crossing between lineage (A) and lineage (B) are analyzed by the method described in Example 13 and the report by Tomizuka et al. (Proc. Natl. Acad. Sci. USA., vol. 97, 722-727, 2000). All the offspring mice obtained by this crossing are heterozygotes for the endogenous antibody heavy chain deficiency and κ chain deficiency, and individuals carrying ΔHAC (or ΔΔHAC) and individuals carrying fragment W23 are selected therefrom and crossed with further obtain offsprings. Individuals (lineage (D)) which are homozygotes for the endogenous antibody heavy chain deficiency and the κ chain deficiency and simultaneously carry ΔHAC (or ΔΔHAC) and fragment W23 are finally selected.

[0139] In lineage (D), expression of the human immunoglobulin heavy chain, κ chain, and λ chain are confirmed by the method described in the report by Tomizuka et al. (Proc. Natl. Acad. Sci. USA., vol. 97, 722-727, 2000) and (WO 98/37757).

[Example 15] Construction of Mouse Lineage Carrying ΔHAC and Having Alleles of Both Endogenous Ig Heavy Chain and κ Chain Genes Destructed

[0140] TC (ΔHAC) produced in Example 13 was back crossed with the endogenous Ig heavy chain and κ chain knock-out mouse lineage described in the report by Tomizuka et al. (Proc. Natl. Acad. Sci. USA., vol. 97, 722-727, 2000). The obtained individual mice were analyzed for the genotype by PCR and ELISA (see Example 12 and the report by Tomizuka et al.).

[0141] As a result, individuals which carried ΔHAC and were homozygotes for the endogenous Ig heavy chain knock-out and were homozygotes for the endogenous Igκ chain knock-out were obtained (hereinafter referred to as “TC(ΔHAC)/ΔH/Δκ”).

[0142] In serum of two TC(ΔHAC)ΔH/Δκ individuals (8-week old), expression of the human Ig heavy chain and λ chain proteins was analyzed by ELISA described in the report by Kuroiwa et al. (Nature Biotechnol., 18: 1086-, 2000). As a result, the expression level in each mouse was as follows: human Ig μ chain: 430 μg/ml, Ig γ chain: 180 μg/ml, Ig λ chain: 330 μg/ml; and human Ig μ chain: 720 μg/ml, Ig γ chain: 320 μg/ml, Ig λ chain: 520 μg/ml.

[Example 16] Construction of Mouse Lineage Carrying a Fragment of Human Chromosome 2 Containing ΔHAC and Human Ig κ Chain Gene and Having Alleles of Both Endogenous Ig Heavy Chain and κ Chain Genes Destructed

[0143] Individual mice obtained by crossing between a mouse lineage carrying a fragment of human chromosome 2 (hCF(W23)) containing the human Ig κ chain gene (hereinafter referred to as “TC(W23)/ΔH/Δκ”) as well as the genetic background of the endogenous Ig heavy chain and κ chain knock-out mouse described in the report by Tomizuka et al. (Proc. Natl. Acad. Sci. USA., vol. 97, 722-727, 2000) and TC (ΔHAC)(ΔH/Δκ lineage produced in Example 15 were analyzed for the genotype in the same manner as in Example 15.

[0144] As a result, individuals were obtained which simultaneously carried ΔHAC and hCF(W23) and were homozygotes for the endogenous Ig heavy chain knock-out and homozygotes for the endogenous Ig κ chain knock-out (hereinafter referred to as “TC(ΔHAC)/TC(W23)/ΔH/Δκ”).

[0145] Further, serum of TC(ΔHAC)/TC(W23)/ΔH/Δκ individuals can be analyzed by ELISA as described in the report by Tomizuka et al. (Proc. Natl. Acad. Sci. USA., vol. 97, 722-727, 2000) and the report by Kuriowa et al. (Nature Biotechnol., 18: 1086-, 2000). Thus, expression of the human Ig μ chain, γ chain, λ chain, and κ chain proteins are respectively detected.

[Example 17] Construction of Mouse Lineage Carrying Yeast Artificial Chromosome Containing ΔHAC and Human Ig κ Chain Gene and Having Alleles of Endogenous Both Ig Heavy Chain and κ Chain Genes Destructed

[0146] Individual mice obtained by crossing between a mouse lineage carrying a transgene containing human Ig κ chain gene (KCo5: containing about 40% of the variable region in the human κ light chain gene) as well as the genetic background of the endogenous Ig heavy chain and a chain knock-out mouse described in the report by Fishwild et al. (Nature Biotechnol., 14: 845-851, 1996) [obtained from Medarex, U.S.A., hereinafter referred to as “KCo5/ΔH/Δκ” ] and TC(ΔHAC)/ΔH/ΔK lineage produced in Example 15 were analyzed for the genotype by PCR and ELISA in the same manner as in Example 15.

[0147] As a result, individuals were obtained which simultaneously carried ΔHAC and KCo5 and were homozygotes for the endogenous Ig heavy chain knock-out and homozygotes for the endogenous Ig κ chain knock-out (hereinafter referred to as “TC(ΔHAC)/KCo5/ΔH/Δκ”).

[0148] Microorganisms retaining a yeast artificial chromosome- or a plasmid constituting the transgene KCo5 are deposited at ATCC (U.S.A.). The accession numbers are as follows. Yeast retaining yeast artificial chromosome y17: ATCC No. PTA-3842, Escherichia coli retaining plasmid pKV4: ATCC No. PTA-3843, Escherichia coli retaining plasmid pKCIB: ATCC No. PTA-3844.

[0149] Serum of TC(ΔHAC)/KCo5/ΔH/Δκ individuals was analyzed by ELISA in the same manner as in Example 16, and as a result, human Ig μ chain, γ chain, λ chain, and κ chain proteins were detected. The average values for the γ chain in the assayed 3 individuals were higher than those for the μ chain.

[Example 18] Production of Anti G-CSF Antibody in the Mouse Lineage TC(ΔHAC)/ΔH/Δκ

[0150] Two individual TC(ΔHAC)/ΔA/Δκ mice produced in Example 15 were immunized with human G-CSF. TiterMaxGold (CytRx) was used as an adjuvant. First, 37.5 μg in total of human G-CSF was immunized subcutaneously in three separate sites. Then, the second and third times, 10 μg in total was immunized subcutaneously in three separate sites as with the initial immunization, 14 days and 38 days after the initial immunization respectively. The final immunization 48 days after the initial immunization was carried out by intravenous injection of 10 μg of G-CSF without any adjuvant. Blood sampling was carried out 3 days after the final immunization and the values for the anti G-CSF human Ig G antibody and for the human Ig λ antibody in serum were measured by ELISA as described in the report by Kuroiwa-et al. (Nature Biotechnol., 18: 1086-, 2000). As a result, an increase in the values for the anti human G-CSF human Ig G antibody and for the human Ig λ antibody was observed in both of the individuals.

[0151] Further, by screening by ELISA a hybridoma obtained by fusion between a spleen cell of the immunized individual mouse and a mouse myeloma cell (Ando, Chiba, “Tan-kurohn Koutai Jikken Sousa Nyuumon (Monoclonal Antibody Experimentation and Manipulation Introduction),” Kodansha Scientific, 1991), a hybridoma producing a complete human monoclonal antibody comprising human Ig heavy chain and light chain λ can be obtained.

[Example 19] Production of Anti G-CSF Antibody in Mouse Lineage TC(ΔHAC)/TC(W23)/ΔH/Δκ

[0152] The individual mouse TC(ΔHAC)/TC(W23)/ΔH/Δκ produced in Example 16 was immunized with human G-CSF in the same manner as in Example 18. The values for the anti G-CSF human Ig G antibody, human Ig λ antibody, and human Ig κ antibody in serum of this mouse are measured by ELISA to confirm an increase in the values for the anti human G-CSF human Ig G antibody, human Ig λ antibody, and human Ig κ antibody.

[0153] In the same manner as in Example 18, a hybridoma producing a complete human monoclonal antibody comprising the human Ig heavy chain and λ light chain or κ light chain can be further obtained by fusion between a spleen cell of the immunized individual mouse and a mouse myeloma cell.

[Example 20] Production of Anti G-CSF Antibody in Mouse Lineage TC(ΔHAC)/KCo5/ΔH/Δκ

[0154] The individual mouse TC(ΔHAC)/TC(W23)/ΔH/Δκ produced in Example 17 was immunized with human G-CSF in the same manner as in Example 18 to measure the values for the anti G-CSF human Ig G antibody, human Ig λ antibody, and human Ig κ antibody in serum by ELISA. As a result, an increase in the values for the anti human G-CSF human Ig G antibody, human Ig λ antibody, and human Ig κ antibody was confirmed.

[0155] In the same manner as in Example 18, a hybridoma producing a complete human monoclonal antibody comprising the human Ig heavy chain and the λ light chain or κ light chain was further obtained by fusion between a spleen cell of the immunized individual mouse and a mouse myeloma cell.

[0156] All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

INDUSTRIAL APPLICABILITY

[0157] The present invention provides a human artificial chromosome which retains a total region of the human antibody heavy chain and λ light chain genes and is genetically transmissible to the next generation with high efficiency. The present invention also provides a non-human animal which genetically transmits the human artificial chromosome to the next generation with high efficiency and an offspring thereof. Further, the present invention enables the production of a human antibody.

FREE TEXT OF SEQUENCE LISTING

[0158] SEQ ID NO: 1, description of artificial sequence: primer
SEQ ID NO: 2; description of artificial sequence: primer
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SEQ ID NO: 16; description of artificial sequence: primer