INTRASPECIFIC CELL HYBRIDS FOR MAPPING ABBERANT GENES

我们的目标是开发一种利用基因 种内体细胞杂交中的互补使 一些人类遗传缺陷的染色体图谱 不能由当前过程映射。A罗伯茨综合征 细胞遗传学异常可通过基因纠正的细胞系 在这一发展过程中将研究互补性。 程序。罗伯茨综合征是一种罕见的常染色体隐性遗传 产生严重对称肢体的遗传缺陷 眼球缩小、眼球突出、眼球过长和阴蒂或 阴茎增大。这些患者中的大多数还患有 不寻常的染色体异常。这种异常现象被称为RS 效果，是可见的，因为着丝粒过早分离 一些染色体的突变，并被证明是由于 C带正异色区的普遍排斥力 姐妹染色单体。而这种细胞遗传学缺陷不可能 通过与正常的人类或小鼠细胞共培养而得到纠正， 我们实验室通过融合罗伯茨夫妇纠正了这一错误 一种小鼠细胞系。这一修正被观察到 在种间体细胞杂交中表明这一点 缺陷可以通过正常基因的存在来纠正 产品。我们将利用这种表型校正作为一种工具 在确定罗伯茨夫妇的染色体分配时 综合症基因。为了实现这一点，正常的人类细胞将被 将pSV2-neo转化为显性 筛选系统(Geneticin抗性)用于分离 杂交细胞。携带该质粒的单个染色体将被 通过微细胞融合转移到罗伯茨综合症细胞。 由此产生的杂交将被进行细胞遗传学分析，以 识别被转移的染色体，它应该显示为 三体，并确定细胞遗传学异常是否已经 已更正。通过对一系列这样的混合动力车的分析， 我们将确定一致的染色体 与异常的纠正有关。这种方法 在绘制任何遗传性疾病的图谱时应该特别有用， 无论它是以染色体、生化或 形态异常，可在细胞培养中检测到 但其异常基因产物尚不清楚。

Our objective is to develop a procedure utilizing genetic complementation in intraspecific somatic cell hybrids to enable chromosomal mapping of a number of human genetic defects which cannot be mapped by current procedures. A Roberts Syndrome cell line with a cytogenetic anomaly correctable by genetic complementation will be studied in the development of this procedure. Roberts Syndrome is a rare autosomal recessive genetic defect which produces severe symmetrical limb reduction, exophthalmos, ocular hypertelorism and clitoral or penile enlargement. A majority of these patients also have an unusual chromosomal anomaly. This anomaly, termed the RS effect, is visible as a premature separation of the centromeres of some of the chromosomes and has been shown to be due to a general repulsion of C-band positive heterochromatic regions in sister chromatids. While this cytogenetic defect cannot be corrected by co-cultivation with normal human or murine cells, it has been corrected in our laboratory by fusing the Roberts Syndrome line to a murine cell line. This correction observed in interspecific somatic cell hybrids indicates that this defect can be corrected by the presence of a normal gene product. We will utilize this phenotypic correction as a tool in determining the chromosomal assignment of the Roberts Syndrome locus. To achieve this, normal human cells will be transformed with the plasmid pSV2-neo to provide a dominant selection system (Geneticin resistance) for the isolation of hybrid cells. Single chromosomes carrying the plasmid will be transferred to Roberts Syndrome cells via microcell fusion. The resulting hybrids will be analyzed cytogenetically to identify the transferred chromosome, which should appear as a trisomy, and to determine if the cytogenetic anomaly has been corrected. Through the analysis of a series of such hybrids, we will determine the chromosome which is consistantly associated with the correction of the anomaly. This approach should be especially useful in mapping any genetic disorder, whether it appears as a chromosomal, biochemical or morphological anomaly, which can be detected in cell culture but for which the abnormal gene product is unknown.