Castrop J, Verbeek S, Hofhuis F, Clevers H
Department of Immunology, University Hospital, Utrecht, The Netherlands.
Immunobiology. 1995 Jul;193(2-4):281-7. doi: 10.1016/S0171-2985(11)80556-5.
Molecular events that underlie the well-defined phenotypic changes of the differentiating thymocyte are poorly understood. A candidate gene to control thymocyte differentiation, T cell factor-1 (TCF-1)* encodes a DNA-binding protein. Its mRNA expression pattern is complex during embryogenesis, yet restricted to lymphocytes postnatally. Expression studies on TCF-1 protein have been hampered by the difficulty to raise antibodies due to extreme evolutionary conservation. TCF-1 knock-out mice, generated recently in our laboratory, have strongly decreased numbers of thymocytes, but are otherwise normal. We have used these mice to generate anti-TCF-1 antibodies. By immunization with a recombinant fusion protein, we show that TCF-1 knock-out mice readily yield antiserum titers against human and mouse TCF-1 protein. Wild-type littermates remain unresponsive to TCF-1 while they mount a high-titer antibody response to the fusion protein, Maltose Binding Protein (MBP). Subsequently, TCF-1-specific hybridomas could be prepared from the spleens of immunized knock-out mice. This study illustrates the almost complete tolerance of mice for human TCF-1 and demonstrates that this tolerance is readily broken by gene knock-out. Furthermore, the usefulness of knock-out mice for the generation of monoclonal antibodies against the gene product of interest is underscored.
对分化中的胸腺细胞明确的表型变化背后的分子事件了解甚少。作为控制胸腺细胞分化的一个候选基因，T细胞因子-1（TCF-1）*编码一种DNA结合蛋白。其mRNA在胚胎发育过程中的表达模式很复杂，但出生后仅限于淋巴细胞表达。由于其高度的进化保守性，很难制备抗体，这阻碍了对TCF-1蛋白的表达研究。我们实验室最近培育出了TCF-1基因敲除小鼠，其胸腺细胞数量大幅减少，但其他方面正常。我们利用这些小鼠制备了抗TCF-1抗体。通过用重组融合蛋白进行免疫，我们发现TCF-1基因敲除小鼠很容易产生针对人和小鼠TCF-1蛋白的抗血清效价。野生型同窝小鼠对TCF-1无反应，但对融合蛋白麦芽糖结合蛋白（MBP）能产生高效价抗体反应。随后，可以从免疫后的基因敲除小鼠脾脏中制备出TCF-1特异性杂交瘤。这项研究说明了小鼠对人TCF-1几乎完全耐受，并证明这种耐受很容易因基因敲除而被打破。此外，强调了基因敲除小鼠在产生针对感兴趣基因产物的单克隆抗体方面的实用性。
