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1.
D Kozbor P Tripputi J C Roder C M Croce 《Journal of immunology (Baltimore, Md. : 1950)》1984,133(6):3001-3005
We produced somatic cell hybrids between human myeloma cells and a lymphoblastoid cell line that is hypoxanthine phosphoribosyl transferase-deficient and ouabain-resistant. These hybrids were phenotypically similar to the human myeloma parental cells and grew as well as the human lymphoblastoid parental cells. After counterselection in 6-thioguanine, mutants that were 6-thioguanine-and ouabain-resistant were obtained, one of which was used as a fusion partner with lymphoblastoid B cells that produce anti-tetanus toxoid (TT) antibodies. These hybrids secreted human anti-TT monoclonal antibodies in much larger amounts than the parental lymphoblastoid cells, and were stable for a period of over 10 mo until the present time. Thus, by hybridizing plasmacytomas with lymphoblastoid cells, we constructed a fusion partner that secretes large amounts of immunoglobulin (Ig), grows at a fast rate, has a high fusion frequency, and supports the production of monoclonal antibodies over long periods of time. Moreover, anti-TT antibody-producing hybrids have been grown as solid tumors in irradiated BALB/c nude mice and then adopted to ascites growth, producing 1 to 8 mg of human immunoglobulin per 1 ml of ascites fluid. 相似文献
2.
Somatic cell hybridization techniques have allowed the preparation of interspecies hybrids that express the features of both parental cell lines. We have studied hybrids made with human myeloma cells fused to a continuous mouse myeloma cell line. In the present study we analyzed the kinetics of leucine influx and efflux in Ig producer and nonproducer hybrids. We found no statistical difference in amino acid influx; however, the rates of efflux were markedly increased in nonproducer hybrids as compared to the producers. The producer cells were tested further in puromycin known to inhibit protein synthesis. Under these conditions amino acid influx was not altered, but efflux was markedly increased resembling the findings in nonproducers. We conclude that hybrids that synthesize human immunoglobulins show decreased efflux of labeled leucine and this effect can be abolished by inhibition of protein synthesis. This difference in the efflux rate appears to be a consequence of immunoglobulin synthesis, rather than a component of a control mechanism of Ig synthesis. 相似文献
3.
4.
In contrast to the mouse immunoglobulin heavy chain and kappa light chain genes, very little is known about the regulation of expression of the immunoglobulin lambda light chain locus. To identify elements responsible for lambda gene regulation we mapped DNaseI hypersensitive sites associated with a functionally rearranged lambda 1 gene in nuclei from the myeloma cell line J558L. Tissue-specific hypersensitive sites were identified 2.3 to 2.5 kb upstream of the CAP site of both the lambda 1 gene and the unrearranged variable (V) lambda 2 gene segments. DNA sequences flanking the lambda 1 gene were isolated and tested for their influence on expression of the lambda 1 gene after transfection into myeloma cells and after injection into fertilized mouse eggs. Two enhancer elements were identified downstream of the lambda 1 gene. A proximal element (located 4 to 10 kb 3' of the gene) enhanced expression of a lambda 1 gene in stable myeloma cell transfectants but had no effect on the expression of a heterologous reporter gene in transient assays. A second, distal element, located approximately 30 kb 3' of the gene, enhanced heterologous expression in J558L cells expressing a lambda gene but not in a non-lambda myeloma cell line (SP2/0-Ag14). Co-injection of cosmids containing the lambda 1 gene and both the proximal and distal downstream elements into fertilized mouse eggs resulted in high-level expression of the lambda 1 transgene in B cells of transgenic mice. The identification of these lambda regulatory elements, in addition to contributing to an understanding of lambda gene regulation per se, will facilitate the study of the regulation of differential expression of kappa and lambda light chain genes in the immune system. 相似文献
5.
Expression of human T antigens in interspecies hybridomas 总被引:2,自引:0,他引:2
Interspecies hybrids were constructed by fusing normal human male peripheral blood mononuclear cells with BW5147, a HGPRT- thymoma derived from an AKR mouse. Hybrid cells were selected in HAT media in culture dishes containing 1 X 10(7) human red blood cells. Twelve weeks after fusion, hybridomas were diluted to 10-15 cells/well and characterized for their expression of the human immune cell surface antigens HLA-DR, T3, T4, and T8 using fluorescent microscopy and cytographic analysis. More than 70% of the hybrid colonies expressed human T-cell surface antigens. Moreover the specific human repetitive DNA (ALU) bound to DNA sequences isolated from the hybridomas after Southern transfers. However, the same hybrids did not have a statistically significant increase in their chromosome number when compared to the mouse parent cell line. Several of the hybridomas produced a soluble factor capable of stimulating the growth of the IL-2 restricted murine cell line CTLL-2 and supported DNA synthesis in human peripheral T-cell populations. Panning experiments demonstrated that the IL-2 producing hybridomas could be enriched by selecting for the human T-cell surface antigen T3. The results presented here indicate that mouse X human hybridomas which express a broad range of human lymphocyte markers can be constructed and maintained in continuous culture for extended periods of time. It also appears that the T3-Ti receptor complex mediates the proliferation of T cells through the T3 molecules linkage to the secretion and/or production of IL-2. The usefulness of interspecific T-cell hybrids as an immunogenetic research tool as well as the significance of the mapping data are discussed. 相似文献
6.
We selected an 8-azaguanine-resistant variant of a human myeloma cell line (RPMI 8226) by cloning the parental cells on a feeder layer of mouse spleen cells in the presence of increasing concentrations of 8-azaguanine. Culture media and cellfree extracts of both the parental and variant (8226 AR/NIP4-1) cell lines were assayed for production of immunoglobulin heavy and light chains by double immunodiffusion and for lambda-chain by radioimmunoassay. Secretion of free lambda-chain by the parental cell line was confirmed. In contrast, no immunoglobulin heavy or light chains were detected in culture medium of the variant cell line by either immunodiffusion or radioimmunoassay. No intracellular lambda-chain could be detected in the variant cells by radioimmunoassay of cellfree extracts or by immunofluorescence of fixed cells. Hybridomas were produced by fusion of 8226AR/NIP4-1 cells with lymphocytes from a mesenteric lymph node recovered at surgery from a hypertransfused renal transplant recipient. Twenty hybrid culture supernatants were assayed for immunoglobulin by double immunodiffusion, and 15 contained either IgG (lambda) or IgG (kappa). None produced IgM or IgA. An IgG (kappa)-producing hybridoma was shown by immunofluorescence not to express lambda-chain. A second fusion between the variant cell line and spleen cells from a renal transplant patient produced a stable hybridoma secreting IgM (lambda) antibody specific for the I antigen. 相似文献
7.
Kazunari Takahashi Satoshi Tereda Hiroshi Ueda Fusao Makishima Eiji Suzuki 《Cytotechnology》1994,15(1-3):57-64
Suppression of proliferation of cells which contain stable or stabilized mRNA coded for a protein to be produced, a partial mimic of cell differentiation, was examined for enhancing protein production by cultured mammalian cells. Hybridoma 2E3 cells which were adapted to be interleukin-6 sensitively growth-suppressed accumulated the mRNA of IgG1 which is reported stable, and IgG1 production rate increased as a result when their growth was suppressed with interleukin-6. A myeloma cell line was similarly adapted; the obtained myeloma cells can be used as host cells for enhancing production of exogenous proteins by suppressing growth with interleukin-6. Temperature-sensitively growth-suppressible mutants of mouse mammary carcinoma FM3A were transfected with cDNA of IgM 1 chain and cultured at nonpermissive temperature to enhance production of 1. Addition of various growth-suppressive reagents to culture medium was studied for finding methods suitable for suppressing growth while maintaining high cell viability. Caffeine yielded the best results among these reagents. Deprivation of various growth-supporting components in culture medium was also tested; simultaneous deprivation of insulin and transferrin viably suppressed growth of hybridoma 2E3 cells, resulting in enhanced antibody productivity.Abbreviations IL6
recombinant human interleukin-6
- TGF-
recombinant human TGF-1
- X63.653-P3X63
Ag8.653 myeloma 相似文献
8.
M11D杂交瘤细胞株是由人胎盘细胞膜纯化所得胰岛素受体免疫BALB/C小鼠后,取其脾细胞与同系小鼠骨髓瘤细胞株NS-1细胞融合所得。该杂交瘤细胞分泌的抗体经ELISA及放射免疫沉淀法证实为胰岛素受体特异的单克隆抗体。该抗体经Protein A-Sepharose亲和层析分离、纯化,SDS-聚丙烯酰胺梯度凝胶电泳鉴定得分子量分别为53000及23000的两条区带,免疫双扩证明为IgGl。该抗体特异地沉淀125Ⅰ-人胎盘细胞膜胰岛素受体,沉淀经SDS-聚丙烯酰胺凝胶电泳后放射自显影得分子量为135000的特异显影带,与胰岛素受体α亚基分子量相同,说明M11D为抗胰岛素受体α亚基的单克隆抗体。 相似文献
9.
Untranslated immunoglobulin kappa light chain mRNA in a lambda light chain-producing mouse myeloma, MOPC104E 总被引:2,自引:0,他引:2
T Kataoka M Ono M Kawakami Y Ikawa M Aida Y Mano T Honjo 《The Journal of biological chemistry》1980,255(11):5291-5295
Fourteen clones were isolated in culture from a mouse myeloma, MOPC104E. All clones had kappa and lambda types of light chain mRNAs in approximately equimolar quantity as assayed by hybridization with specific complementary DNA (cDNA). However, the myeloma produces and secretes only lambda-type light chain protein. Both kappa- and lambda-type mRNAs in these clones were indistinguishable from kappa- and lambda-type mRNAs of other myelomas with respect to (a) adsorption to oligo-(dT) cellulose, (b) molecular size (12.6 S), and (c) thermal stability of the hybrids formed with corresponding cDNA. The kappa chain mRNA of MOPC104E cells, however, was translated very inefficiently both in vivo and in vitro, whereas the lambda chain mRNA was translated efficiently. These results indicate that each cell of MOPC104E myeloma synthesizes a crippled kappa chain mRNA in addition to a normal lambda chain mRNA. 相似文献
10.
Characterization of monoclonal antibodies against heavy and light chains of flounder (Paralichthys olivaceus) immunoglobulin 总被引:2,自引:0,他引:2
Jang HN Woo JK Cho YH Kyong SB Choi SH 《Journal of biochemistry and molecular biology》2004,37(3):314-319
Flounder (Paralichthys olivaceus) Immunoglobulins (Igs) were purified from the serum of mouse IgG-immunized flounder by using affinity chromatography. Under denaturing conditions in SDS-PAGE, the flounder Igs appeared to be composed of 2 heavy (H) chains (72 and 77 kDa) and two light (L) chains (26 and 28 kDa). Monoclonal antibodies (MAbs) were produced by the fusion of myeloma cells (SP2/0) with Balb/c mouse spleen cells that were previously sensitized against affinity-purified flounder Igs. In a Western blot analysis, the produced MAbs, FIM511, FIM519, and FIM562 recognized both the 72 and 77 kDa H chains, 26 kDa, and 28 kDa L chain, respectively. Mouse antiserum against flounder Igs reacted more strongly with the L chain of 28 kDa than with 26 kDa, suggesting that the 28 kDa molecule is more immunogenic than the 26 kDa L chain molecule. In a FACS analysis, the ratios of the Ig+ cell population in the flounder head kidney and spleen cells were 49% and 24%, respectively. Unexpectedly, however, the ratios of the Ig+ B-like cell population in the flounder were not significantly augmented, even after the immunization of an immunogenic antigen. This suggests that the humoral immune response in fish could be considerably different from that in mammals. The produced MAbs in this study would be useful in characterizing flounder Ig+ B-like cells and in developing flounder Ig detecting an immunoassay system. 相似文献
11.
H J Garchon A Blancher F Champommier A Blanchard de Vaucouleurs J F Bach J Hainaut 《Revue fran?aise de transfusion et immuno-hématologie》1983,26(2):147-151
Peripheral blood lymphocytes from healthy people recently immunized against tetanus toxoid (TT) were fused with human malignant B-cell lines or mouse myeloma cells (X63 Ag8.653) in an attempt to establish stable B cell hybridomas secreting anti TT antibodies. Human-human fusion experiments were not successful. In contrast, the five heterospecific fusion experiments yielded between 60 and 100% of wells that contained growing hybrids. Five of these hybrids repeatedly secreted anti-TT antibodies. One of the hybrids was cloned and secreted 10-20 micrograms/ml of human IgM, lambda anti-TT antibody. Heterospecific hybridization thus appears as an interesting method to obtain human monoclonal antibodies, allowing the study of their properties. 相似文献
12.
The plasmacytoma J558L lacks constitutively active NF-kappa B and is deficient in early response gene activation.
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In mature B cells the nuclear factor NF-kappa B which binds within the kappa enhancer is constitutively present in the nucleus. However, the lambda light chain producing myeloma J558L has been found to lack constitutively functional NF-kappa B. Deoxycholate released functional NF-kappa B from cytoplasmic extracts and functional NF-kappa B was present in J558L following cycloheximide but not phorbol ester treatment. J558L was also unable to respond to phorbol ester stimulation with synthesis of mRNA from the early response gene TIS11. J558L differs from S107, another myeloma which was found to be deficient in the synthesis of NF-kappa B but not in the activation of TIS11. Somatic cell hybrids were used to further define the defect in J558L; hybrids were made with the myelomas S107 and S194 and the pre-B cell line 70Z/3. In general, complementation of the defect in J558L was observed; however there was not a direct correlation between the levels of TIS11 mRNA and NF-kappa B expression in the somatic cell hybrids, suggesting that the pathways of activation of these genes, while possibly sharing common elements, are not identical. The defect in J558L was surprising given that it has frequently been used for the expression of transfected light chain genes. 相似文献
13.
A hybridoma producing human monoclonal antibody specific for glycoprotein 120 kDa of human immunodeficiency virus (HIV-1 总被引:1,自引:0,他引:1
D Lake T Sugano Y Matsumoto Y Masuho E A Petersen P Feorino E M Hersh 《Life sciences》1989,45(16):iii-iix
A stable hybridoma producing anti-HIV human monoclonal antibody (HMCA) was generated by fusing CD3-depleted human splenic lymphocytes from an HIV sero-positive donor with the mouse myeloma cell line P3x63AgU1. The resultant hybridoma has been secreting IgG1, lambda chain for over nine months at a rate of 2.5 micrograms/10(6)cells/day. The HMCA shows specific reactivity in ELISA using HIV-infected cell lysates. Immunofluorescence tests have indicated that this HMCA binds specifically to the surface of H9 and C3 HIV/HTLVIIIb infected cells, HIV/N1T infected CEM cells and to MoT cells infected with an HIV clinical isolate. Western blotting revealed recognition of glycoproteins 120 and 160 kDa of HIV by the HMCA. Although this HMCA demonstrated no neutralizing activity, the production of an anti-HIV HMCA specific for glycoprotein 120 kDa indicates the possibility that a neutralizing HMCA can be developed as further fusions with lymph nodes and spleens from HIV positive donors are performed. 相似文献
14.
Human myeloma cell line RPMI 8226 synthesizes and secretes a lambda immunoglobulin light chain. Total cellular RNA, obtained from cells grown in culture, was used for the isolation of poly(A)-containing mRNA by oligo(dT)-cellulose chromatography. The poly(A)-containing mRNA was translated in an Ehrlich ascites extract. Immunoprecipitation of the cell-free products showed that a polypeptide chain antigenically related to human lambda chain was synthesized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the cell-free product was larger than the secreted light chain. On tryptic digestion the cell-free product and the secreted light chain exhibited essentially identical peptide patterns except for an additional peptide derived from the cell-free product. We conclude that the lambda mRNA from this human myeloma cell line codes for a precursor to the secreted lambda chain as described for immunoglobulin mRNAs from murine plasmacytomas. 相似文献
15.
The production of both protein and steroid hormones was studied utilizing somatic cell hybrids formed with human choriocarcinoma cells. The human JEG-3 cell line produced the species and organ-specific hormone human chorionic gonadotropin (hCG), the steroid hormone progesterone, and converted 19-carbon steroids to estrogens. Hybrids formed with human VA-2 cells, mouse Cl 1D cells and mouse 3T3-4EF cells had detectable hCG synthesis in 20 of 41 total clones. There was no detectable progesterone or 19-carbon aromatization to estrogens in any hybrids. These data demonstrate that the differentiated function of human protein hormone production can be retained in inter- and intra-specific somatic cell hybrids. These results also suggest that protein hormone production can occur independently of steroid production in these cells of placental origin. 相似文献
16.
J Schwaber 《Somatic cell genetics》1977,3(3):295-302
Fusion of unfractionated human lymphocytes with mouse myeloma cells resulted in proliferating hybrid colonies, almost all producting human Ig. We examined whether this high frequency of Ig production was the result of selective formation of human B lymphocyte-mouse myeloma hybrids, rather than induction of Ig genes in T lymphocytes. Unfractionated peripheral lymphocytes and B lymphocytes from patients with the common variable form of agammaglobulinemia formed proliferating somatic cell hybrid colonies. In contrast, peripheral lymphocytes from a patient with agammaglobulinema who lacked B lymphocytes, as well as albumin gradient fractions of peripheral blood which do not contain B lymphocytes, failed to produce somatic cell hybrids with three different myeloma parent cell lines. B, T, and precursor lymphocytes all had Sendai virus receptors, as witnessed by viral agglutination. We conclude that fusion of human lymphocytes with mouse myeloma cells results in selective hybrid formation, rather than activation of Ig genes in disparate cell types. Only B lymphocyte-mouse myeloma heterokaryons form hybrid cells. 相似文献
17.
Integration of Ecogpt and SV40 early region sequences into human chromosome 17: a dominant selection system in whole cell and microcell human-mouse hybrids 总被引:7,自引:1,他引:6
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Tunnacliffe A Parkar M Povey S Bengtsson BO Stanley K Solomon E Goodfellow P 《The EMBO journal》1983,2(9):1577-1584
The dominant selectable gene, Ecogpt, has been introduced, by the calcium phosphate precipitation technique, into normal human fibroblasts, along with the SV40 early region genes. In one transfectant clone, integration of these sequences into human chromosome 17 was demonstrated by the construction of human-mouse somatic cell hybrids, selected for by growth in medium containing mycophenolic acid and xanthine. A whole cell hybrid, made between the human transfectant and a mouse L cell, was used as donor of the Ecogpt-carrying human chromosome 17 to 'tribrids' growing in suspension, made by whole cell fusion between a mouse thymoma cell line, and to microcell hybrids made with a mouse teratocarcinoma cell line. Two tribrids contained karyotypically normal human chromosomes 17 and a small number of other human chromosomes, while a third tribrid had a portion of the long arm of chromosome 17 translocated to mouse as its only human genetic material. Two independent microcell hybrids contained a normal chromosome 17 and no other human chromosome on a mouse teratocarcinoma background. These experiments demonstrate the ability to construct human-mouse somatic cell hybrids using a dominant selection system. By applying this approach it should be possible to select for a wide range of different human chromosomes in whole cell and microcell hybrids. In particular, transfer of single human chromosomes to mouse teratocarcinoma cells will allow examination of developmentally regulated human gene sequences after differentiation of such hybrids. 相似文献
18.
Esterase gene expression in Chinese hamster and mouse lymphoma hybrids isolated under nonselective pressure 总被引:1,自引:0,他引:1
Hybrids between a fibroblastic Chinese hamster cell line (CH23) and a mouse lymphoma cell line (P388F36) were produced and isolated by a simple new method without using selective media and avoiding contact with the parental cells. The chromosomal situation in the two hybrid types (PCM and PCS) isolated suggested that growth on glass surface (PCM) or in suspension (PCS) depended on the number of hamster and mouse chromosomes which existed in the hybrids. Chromosomal stability in hybrids grown as monolayers (PCM) was reached at a stage in which two to four mouse chromosomes coexisted with no fewer than 19 hamster chromosomes. In a study of gene linkage utilizing clones of this hybrid population, five out of nine genes regulating the synthesis of different esterases in the mouse cells used were found to be unlinked. 相似文献
19.
Hybrid cells were prepared by fusin an immunoglobulin-secreting mouse myeloma lin e (B cell) with an allogenic T-cell lymphoma which expresses the surface antigen Thy 1. The resulting hybrids expressed H2 antigens of both parental cells and secreted the immunoblobulin of the myeloma parent but did not express the Thy 1 antigen of the lymphoma parent. Twenty-one hybrids were formed from fusion of the same myeloma line with TNP-SRBC-primed spleen cells. Most of the hybrid lines exhibited characteristics expected for the fusion of the myeloma to B lymphocytes. No hybrids between the myeloma line and spleen T cells were identified as none of the hybrids expressed the T-cell-specific antigen Thy 1. We discuss possible reasons for failure to produce hybrids with T-cell characteristics in these types of fusion. 相似文献
20.
Arrangement of lambda light chain genes in mutant clones of the MOPC 315 mouse myeloma cells 总被引:2,自引:0,他引:2
N Hozumi G Wu H Murialdo R Baumal T Mosmann L Winberry A Marks 《Journal of immunology (Baltimore, Md. : 1950)》1982,129(1):260-266
The synthesis of lambda light chains and the arrangement of the lambda-chain genes was examined in cells of the mouse myeloma MOPC 315, which is an alpha lambda 2 producer, and in several mutants derived from it. The mutants produce lambda 2 chains only (MOPC 315.26, MOPC 315.34, and MOPC 315.37) or fail to produce alpha and lambda 2 chains (MOPC 315.25 and MOPC 315.36). Messenger RNA from the lambda 2 chain-producing cells directed the synthesis of a lambda 2 chain precursor and a fragment of the lambda 1 chain (lambda 1 F) in a wheat embryo cellfree system, whereas mRNA from the cells that do not produce lambda 2 chains directed the synthesis of lambda 1 F only. DNA from the parental MOPC 315 cells and from the lambda 2 chain-producing cells contained discrete EcoRI restriction fragments coding for rearranged lambda 1 and lambda 23 chain genes and their respective germ-line V and J-C regions. DNA from the no-Ig-producing cells contained fragments coding for the rearranged lambda 1 chain gene and the germ-line V lambda 2 region, but it lacked the sequences coding for the rearranged lambda 2 chain gene and the germ-line V lambda 1 and J-C lambda 1 regions. These results suggest that rearrangements of the lambda 1 and lambda 2 chain genes occur on different chromosomes in MOPC 315 cells and imply that rearrangements of the lambda 1 and lambda 2 chain genes on the same chromosome may be mutually exclusive. 相似文献