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首页> 美国卫生研究院文献>Molecular and Cellular Biology >Expression of T-cell receptor alpha-chain genes in transgenic mice.
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Expression of T-cell receptor alpha-chain genes in transgenic mice.

机译：T细胞受体α链基因在转基因小鼠中的表达。

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To examine the influences responsible for shaping the T-cell repertoire in vivo, we have introduced T-cell receptors of defined specificity into mice. In this report, we analyze transgenic mice carrying a T-cell receptor alpha-chain gene from a pigeon cytochrome c-reactive T-cell line. A variant of this construct, which has the immunoglobulin heavy-chain enhancer inserted into the JC intron, was also introduced into mice. Addition of the enhancer increased the steady-state level of transgene-encoded mRNA three- to fivefold in cultured T cells, leading to a two- to threefold increase in surface expression. In vivo, the difference between these two constructs was even more significant, increasing the number of transgene-positive cells from approximately 5 to 70% and the T-cell receptor surface density two- to threefold. Surprisingly, while surface expression of either type of transgene was limited to T cells, we found little tissue specificity with respect to transcription. In T cells expressing the alpha chain from the enhancer-containing construct, immunoprecipitation with a 2B4 alpha-specific monoclonal antibody revealed the expected disulfide-linked dimer. Costaining of these T cells with the 2B4 alpha-specific monoclonal antibody versus anti-CD3 indicated that expression of the transgene-encoded alpha chain precludes expression of endogenous alpha chains on the majority of cells; in contrast, 2B4 alpha-chain expression from the construct lacking the enhancer is inefficient at suppressing endogenous alpha-chain expression. In mice of the enhancer lineage, Southern blot analysis indicated suppression of endogenous alpha-chain rearrangements in T-cell populations, consistent with the observed allelic exclusion at the cellular level. Interestingly, newborn, but not adult, mice of this lineage also showed an increase in retention of unrearranged delta-chain loci in thymocyte DNA, presumably resulting from the suppression of alpha-chain rearrangements. This observation indicates that at least a fraction of alpha:beta-positive T cells have never attempted to produce functional delta rearrangements, thus suggesting that alpha:beta and gamma:delta T cells may be derived from different T-cell compartments (at least during the early phases of T-cell differentiation).
机译：为了检查负责体内塑造T细胞库的影响，我们将定义特异性的T细胞受体引入小鼠。在这份报告中，我们分析了从鸽子细胞色素c反应性T细胞系携带T细胞受体α链基因的转基因小鼠。该构建体的变体也已引入小鼠体内，该构建体的免疫球蛋白重链增强剂已插入JC内含子中。增强剂的添加将培养的T细胞中转基因编码的mRNA的稳态水平提高了三到五倍，导致表面表达提高了两到三倍。在体内，这两种构建体之间的差异甚至更大，将转基因阳性细胞的数量从大约5％增加到70％，T细胞受体表面密度增加了两倍至三倍。出人意料的是，尽管任何一种转基因的表面表达都限于T细胞，但我们发现有关转录的组织特异性很小。在表达来自包含增强子的构建体的α链的T细胞中，用2B4α特异性单克隆抗体进行的免疫沉淀显示了预期的二硫键连接的二聚体。用2B4α特异性单克隆抗体和抗CD3对这些T细胞进行染色处理，表明转基因编码的α链的表达排除了大多数细胞上内源性α链的表达。相反，来自缺乏增强子的构建体的2B4α-链表达在抑制内源性α-链表达方面效率低下。在增强子谱系的小鼠中，Southern印迹分析表明抑制了T细胞群体中的内源性α链重排，这与在细胞水平上观察到的等位基因排阻一致。有趣的是，该血统的新生小鼠而非成年小鼠也显示胸腺细胞DNA中未重排的δ链基因座的保留增加，这可能是由于α链重排的抑制所致。该观察结果表明，至少一部分α：β阳性T细胞从未尝试过产生功能性δ重排，因此表明α：β和γ：δT细胞可能源自不同的T细胞区室（至少在T细胞分化的早期阶段）。

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