Cunningham K, Ackerly H, Claflin L, Collins J, Wu P, Ford C, Lansford R, Alt F, Dunnick W A
Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor 48109-0620, USA.
Int Immunol. 1998 Aug;10(8):1027-37. doi: 10.1093/intimm/10.8.1027.
To investigate the regulation of Ig switch recombination, we have constructed mice with a 56 kb VDJmudeltagamma1 transgene. This transgene included an anti-nitrophenyl VDJ segment, Smu, Cmu, Cdelta, Igamma1, Sgamma1, Cgamma1 and the Cgamma1 membrane exons from the murine Igh(a) haplotype. Two founder lines were produced, with very similar characteristics. Transgenic B cells expressed normal amounts of Cmu (which is >95% transgenic), Cdelta and other cell surface markers, and normal amounts of VDJ and Cmu RNA. Gamma1 germline transcription of the transgenes is properly regulated since stable transcripts were not expressed in B cells treated with lipopolysaccharide (LPS) alone, nor in thymus or non-lymphoid tissues, but were expressed after treatment of B cells with LPS + IL-4 or CD40L + IL-4. B cells from both lines of transgenic mice expressed transgenic gamma1a after in vitro culture with CD40L + IL-4, but not after culture with CD40L alone. However, the CD40L + IL-4 induced IgG1 precursor frequency is much lower for VDJmudeltagamma1 transgenic B cells (1:240-760) than for non-transgenic B cells (1:9). Analysis of DNA from transgenic hybridomas indicated that switch recombination can take place in switch (S) regions, but can also take place outside S regions. These results indicate that targeting of switch recombinase to S regions must include regulation beyond the S regions themselves and correct germline transcription. This additional regulation might include cis-acting elements or appropriate spacing or arrangement of the recombining elements.
为了研究Ig转换重组的调控机制，我们构建了带有56 kb VDJmudeltagamma1转基因的小鼠。该转基因包含一个抗硝基苯基VDJ片段、Smu、Cmu、Cdelta、Igamma1、Sgamma1、Cgamma1以及来自小鼠Igh(a)单倍型的Cgamma1膜外显子。产生了两个奠基系，它们具有非常相似的特征。转基因B细胞表达正常量的Cmu（其中>95%为转基因）、Cdelta和其他细胞表面标志物，以及正常量的VDJ和Cmu RNA。转基因的γ1胚系转录受到适当调控，因为在仅用脂多糖（LPS）处理的B细胞中，以及在胸腺或非淋巴组织中均未表达稳定转录本，但在用LPS + IL-4或CD40L + IL-4处理B细胞后表达。来自两个转基因小鼠系的B细胞在与CD40L + IL-4进行体外培养后表达转基因γ1a，但在仅与CD40L培养后不表达。然而，对于VDJmudeltagamma1转基因B细胞，CD40L + IL-4诱导的IgG1前体频率（1:240 - 760）远低于非转基因B细胞（1:9）。对转基因杂交瘤的DNA分析表明，转换重组可发生在转换（S）区域，但也可发生在S区域之外。这些结果表明，将转换重组酶靶向S区域必须包括S区域本身之外的调控以及正确的胚系转录。这种额外的调控可能包括顺式作用元件或重组元件的适当间距或排列。
