2006 Fiscal Year Final Research Report Summary
Molecular evolution system of proteins using a B cell line with spontaneous mutation machinery
| Project/Area Number |
16360414
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biofunction/Bioprocess
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| Research Institution | OKYAMA UNIVERSITY |
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Principal Investigator |
OHMORI Hitoshi Okayama University, Biotechnology, Professor, 大学院自然科学研究科, 教授 (70116440)
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| Project Period (FY) |
2004 – 2006
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| Keywords | antibody / B cell / somatic hypermutation / chicken / protection of infection / protein engineering / lymphoid tissue / molecular evolution technology |
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| Research Abstract |
A chicken B lymphoma line, DT40, hypermutates immunoglobulin (Ig) genes spontaneously during culture. Thus, cultured DT 40 cells constitute a useful Ig library for screening antibodies (Abs) in vitro. To.x desirable Ig mutants by stopping hypermutation or to resume mutation for further improvement of Ab a.nity, activation-induced cytidine deaminase (AID), a key enzyme responsible for the Ig mutation machinery, must be switched on or o.. To this end, we generated a DT40 line whose one AID allele was disrupted, and the other allele was replaced by the loxP-.anked AID construct. In this engineered cell line designated as DT40-SW, AID expression could be switched reversibly by tamoxifen-regulated Cre recombinase. Devices were also introduced to discriminate between the "AID-ON" and the "AID-OFF" cells by GFP expression and puromycin resistance, respectively. Starting from a single DT40-SW cell, Ig gene repertoire was efficiently diversified during culture only when AID expression was on.
We
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generated DT4O lines that bears a gene conversion substrate comprising the green fluorescent protein (GFP) gene as a donor and the blue fluorescent protein (BFP) gene as an acceptor. BFP-expressing cells converted their fluorescence from blue to green when the substrate construct was integrated in the Ig L chain locus. Thus, the gene conversion machinery in DT4O cells will be useful to engineer non-Ig proteins.
To investigate mechanisms underlying germinal center (GC) reaction, we established a cell line designated as pFL that retained major FDC phenotypes from a 3-dimensional culture of mouse lymph node segments. pFL cells proliferated slowly in response to an agonistic anti-lymphotoxin β receptor (LTβR) mAb and TNFα. A more rapidly growing clone, named FL-Y was isolated from a long-term culture of pFL. Analysis of surface markers in these two cell lines revealed the expression of genes that are characteristic of FDCs. Thus, pFL and FL-Y cells may be useful for providing insight into the functional role for FDC in GC Less
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