TY - JOUR
T1 - Convenient plasmid vectors for construction of chimeric mouse/human antibodies
AU - Kameyama, Koh zoh
AU - Imai, Kenji
AU - Itoh, Toshihiro
AU - Taniguchi, Masaru
AU - Miura, Keiji
AU - Kurosawa, Yoshikazu
N1 - Funding Information:
AcknowledgementWs:e thank Drs P. Berg, S. Tonegawa,T . Maniatisa ndM. Potter for providingu s with the materialsD, rs Y. Takagi,I . Ishiguroa nd K. Fujita for encouragemenatn d MS A. Nagataf or preparingt he manuscriptT. his work was supported in part by grants from the Ministry of Education, Science and Culture of Japan and Fujita-Gakuen Health University.
PY - 1989/2/27
Y1 - 1989/2/27
N2 - Chimeric antibodies composed of mouse-derived variable regions and human-derived constant regions have been developed for clinical use. However, construction of chimeric mouse/human genes in expression vectors is time-consuming work. In this study, we developed convenient vectors for construction of chimeric mouse/human antibodies. The protocols are as follows: In mouse hybridomas and B cells, most active VH and Vκ{script} genes can be identified as rearranged bands by Southern hybridization of EcoRI- and HindIII-digested DNAs with JH and Jκ{script} probes, respectively, and such fragments can be isolated in λ-EcoRI and λ-HindIII vectors, respectively. We constructed two plasmids: pSV2-HG1gpt contains human Cγ1 and Ecogpt genes, and only one EcoRI site upstream of the Cγ1 gene; pSV2-HCκ{script}neo contains human Cκ{script} and neo genes, and only one HindIII site upstream of the Cκ{script} gene. An isolated EcoRI fragment containing a VHDHJH gene and a HindIII fragment containing a Vκ{script}Jκ{script} gene are inserted into pSV2-HG1gpt and pSV2-HCκ{script}neo, respectively. Both resulting plasmid DNAs are co-transfected into SP2/0 cell, a non-Ig-secreting mouse myeloma. Transformants are selected by both mycophenolic acid and G418. With this procedure, it takes only 2 months to obtain chimeric antibodies.
AB - Chimeric antibodies composed of mouse-derived variable regions and human-derived constant regions have been developed for clinical use. However, construction of chimeric mouse/human genes in expression vectors is time-consuming work. In this study, we developed convenient vectors for construction of chimeric mouse/human antibodies. The protocols are as follows: In mouse hybridomas and B cells, most active VH and Vκ{script} genes can be identified as rearranged bands by Southern hybridization of EcoRI- and HindIII-digested DNAs with JH and Jκ{script} probes, respectively, and such fragments can be isolated in λ-EcoRI and λ-HindIII vectors, respectively. We constructed two plasmids: pSV2-HG1gpt contains human Cγ1 and Ecogpt genes, and only one EcoRI site upstream of the Cγ1 gene; pSV2-HCκ{script}neo contains human Cκ{script} and neo genes, and only one HindIII site upstream of the Cκ{script} gene. An isolated EcoRI fragment containing a VHDHJH gene and a HindIII fragment containing a Vκ{script}Jκ{script} gene are inserted into pSV2-HG1gpt and pSV2-HCκ{script}neo, respectively. Both resulting plasmid DNAs are co-transfected into SP2/0 cell, a non-Ig-secreting mouse myeloma. Transformants are selected by both mycophenolic acid and G418. With this procedure, it takes only 2 months to obtain chimeric antibodies.
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U2 - 10.1016/0014-5793(89)80550-2
DO - 10.1016/0014-5793(89)80550-2
M3 - Article
C2 - 2920830
AN - SCOPUS:0024548955
SN - 0014-5793
VL - 244
SP - 301
EP - 306
JO - FEBS Letters
JF - FEBS Letters
IS - 2
ER -