ISOLATION OF GENETIC MARKERS FOR INTESTINAL CRYPT CELLS

肠的隐窝和绒毛由复杂的连续的 单细胞层上皮。 多能锚定干细胞定位 在隐窝基部附近产生四个终末分化的 细胞类型，其中大部分迁移到绒毛的尖端， 在三到四天内挤出。 肠腺细胞 是成人中增殖最快的细胞群之一 每个隐窝每天产生约300个细胞。 的 控制细胞快速增殖和分化的机制 在肠道中的作用还不是很清楚。 酪氨酸激酶家族 包括许多生长因子受体和致癌基因， 在多种系统中调节生长和分化。 我们假设酪氨酸激酶也在调节细胞凋亡中起重要作用。 在肠道中的作用。 为了鉴定酪氨酸激酶基因 在隐窝中表达，我们使用聚合酶链反应， 简并寡核苷酸探针对应于两个不变的氨基 在所有酪氨酸激酶中发现的酸性序列基序，以扩增酪氨酸 使用隐窝细胞cDNA作为模板的激酶催化域。 的 克隆了扩增的酪氨酸激酶片段， 表征了 测序已经确定了几个酪氨酸激酶基因 在隐窝中表达，包括14个新基因。 通过检查它们 表达，我们正在确定这些酪氨酸激酶基因中的哪一个是 优先在肠隐窝中转录。 全长cDNA 将分离编码这些酪氨酸激酶的克隆， 表征了 我们将在细胞系中表达这些cDNA， 转基因小鼠，以确定不适当的表达是否会导致 可检测的表型 将产生抗融合的抗体 这些克隆编码的蛋白质，这些将用于开始我们的 酪氨酸激酶蛋白的表征。识别 在肠中表达的酪氨酸激酶，以及分离新的隐窝 细胞酪氨酸激酶cDNA克隆将提供分子工具， 分化程序的遗传学和生物化学剖析， 放置在肠道内。 我们的长期目标是了解 以及在体内和体外对隐窝细胞酪氨酸激酶的调节。

The crypts and villi of the intestine are lined by a complex continuous single cell layer epithelium. Multipotential anchored stem cells located near the base of the crypts give rise to four terminally differentiated cell types, the majority of which migrate to the tips of the villi and are extruded within three to four days. The intestinal crypt cells represent one of the most rapidly proliferating cell populations in adult animals, with each crypt producing about 300 cells per day. The mechanisms controlling the rapid proliferation and cell differentiation in the intestine are not well understood. The family of tyrosine kinases includes a number of growth factor receptors and oncogenes, and these function to regulate growth and differentiation in a variety of systems. We hypothesize that tyrosine kinases also play an important regulatory role in the intestinal crypts. To identify the tyrosine kinase genes expressed in the crypts, we used the polymerase chain reaction primed by degenerate oligonucleotide probes corresponding to two invariant amino acid sequence motifs found in all tyrosine kinases, to amplify tyrosine kinase catalytic domains using crypt cell cDNA as a template. The amplified tyrosine kinase fragments were cloned and 285 clones have been characterized. Sequencing has identified several tyrosine kinase genes expressed in the crypts, including 14 novel genes. By examining their expression, we are determining which of these tyrosine kinase genes is preferentially transcribed in the intestinal crypts. Full length cDNA clones encoding these tyrosine kinases will be isolated and characterized. We will express these cDNAs in cell lines and in transgenic mice, to determine if inappropriate expression results in a detectable phenotype. Antibodies will be generated against fusion proteins encoded by these clones, and these will be used to begin our characterization of the tyrosine kinase proteins. Identification of the tyrosine kinases expressed in the intestine, and isolation of novel crypt cell tyrosine kinase cDNA clones will provide the molecular tools for the genetic and biochemical dissection of differentiation programs taking place in the intestine. Our long-term goal is to understand the function and regulation of crypt cell tyrosine kinases both in vivo and in vitro.