INTRASPECIFIC CELL HYBRIDS FOR MAPPING ABBERANT GENES

我们的目标是开发一种利用遗传 种内体细胞杂种中的互补， 一些人类遗传缺陷的染色体图谱， 当前程序无法映射。 罗伯茨综合征 细胞遗传学异常可通过遗传学纠正的细胞系 将在开发过程中进行研究， procedure. 罗伯茨综合征是一种罕见的常染色体隐性遗传病， 遗传缺陷，导致严重的对称肢体 减少，眼球突出，眼距过宽和阴蒂或 阴茎增大 这些患者中的大多数也有 不寻常的染色体异常 这种异常称为RS 效应，可见为着丝粒的过早分离 的一些染色体，并已被证明是由于 C带正异染色质区的一般排斥 姐妹染色单体 虽然这种细胞遗传学缺陷不能 通过与正常人或鼠细胞共培养来校正， 在我们的实验室里， 综合征细胞系与鼠细胞系的比较。 这一修正观察到 种间体细胞杂交表明， 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工具 决定罗伯茨夫妇的染色体分配 综合症基因座。 为了实现这一目标，正常的人体细胞将 用质粒pSV 2-neo转化以提供显性的 选择系统（遗传霉素抗性）用于分离 杂交细胞 携带质粒的单染色体将 通过微细胞融合转移到罗伯茨综合征细胞中。 将对产生的杂种进行细胞遗传学分析， 识别转移的染色体，它应该显示为 三体性，并确定细胞遗传学异常是否已 更正。 通过对一系列这样的杂交种的分析， 我们将确定染色体， 与异常的纠正相关。 这种方法 应该特别适用于绘制任何遗传疾病， 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 但其异常基因产物未知。

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.