Acquisition of a FACSort for Studying Lymphocyte Differentiation and for Undergraduate Research Training

摘要 我们正在申请资助Becton Dickinson FACsort。 购置这一仪器有三个主要目的。 1)FACsort将广泛用于正在进行的淋巴细胞分化研究。 这些项目涉及每学期6至8名本科生，包括夏季。 2)FACSort将是本科免疫学课程的一个组成部分。 3)使用FACSort的实验将被引入分子生物学课程。 因此，大量的本科生将通过本科课程和研究经验获得荧光激活细胞分选的经验。 西方学院的两个研究项目需要细胞分选。 波洛克博士的研究中心是免疫球蛋白（IG）可变（V）区基因的体细胞突变。 体细胞突变在产生抗体多样性中起重要作用。 我们正在开发一种检测和分离经历免疫球蛋白基因体细胞突变的淋巴细胞的方法。 该检测使用编码融合蛋白的DNA构建体，该融合蛋白由重排的V基因组成，含有终止密码子，与lacZ报告基因连接。 如果体细胞突变使终止密码子恢复，则整个融合蛋白被翻译并表达lacZ。 LacZ表达容易通过荧光测定法检测。 这种构建体正在用于细胞系和制造转基因小鼠。 这些小鼠将被用于询问：1）在哪里可以检测到体细胞突变; 2）在免疫后何时看到体细胞突变; 3）在个体发育期间何时首次发生体细胞突变; 4）哪些生殖中心细胞显示活跃的体细胞突变;以及5）经历体细胞突变的细胞的表型是什么。

ABSTRACT We are requesting funding for a Becton Dickinson FACsort. There are three main objectives for the acquisition of this instrument. 1) The FACsort will be used extensively in ongoing studies on lymphocyte differentiation. These projects involve 6 to 8 undergraduate students every term, including the summer. 2) The FACSort will be an integral part of the undergraduate Immunology course. 3) Experiments using the FACSort will be introduced into the Molecular Biology course. Thus, a large number of undergraduates will gain experience with the fluorescence activated cell sorting through both undergraduate courses and research experience. Two research projects at Occidental College require cell sorting. Dr. Pollock's research centers on somatic mutation of immunoglobulin (Ig) variable (V) region genes. Somatic mutation plays an important role in generating antibody diversity. We are developing an assay to detect and isolate lymphocytes undergoing somatic mutation of immunoglobulin genes. The assay uses a DNA construct encoding a fusion protein composed of a rearranged V gene, containing a stop codon, linked to a lacZ reporter gene. If somatic mutation reverts the stop codon, the entire fusion protein is translated and lacZ expressed. LacZ expression is readily detected by fluorescence assays. This construct is being used in cell lines and to make transgenic mice. These mice will be used to ask: 1) where can somatic mutation be detected; 2) when is somatic mutation seen after immunization; 3) when does somatic mutation first occur during ontogeny; 4) which germinal center cells show active somatic mutation; and 5) what is the phenotype of cells undergoing somatic mutation.