Gene expression in clonogenic versus non-clonogenic in pooled early passage OCI/AML-4 single cells

Identification of genes that regulate clonogenicity of AML cells is hindered by the difficulty of isolating pure populations of cells with defined proliferative abilities. Using early passages of the OCI/AML-4 cell line we sought to determine genes differentially expressed between clonogenic and non-clonogenic cells. We previously shown that OCI/AML-4 clonal siblings display coordinated growth patterns, so that the behaviour of a single cell that is destroyed in the generation of global single cell cDNA may be predicted by the growth patterns of its clonal siblings. Cells were plated in 96 well plates at limiting dilutions. Localized clusters of four cells were identified and micromanipulated such that three of the constituent cells were placed separately into individual microtitre wells containing growth medium and a feeder layer of OP9 cells, while the fourth cell was lysed and processed for global RT-PCR. The cells in each culture well were counted at 2 -3 day intervals until growth stopped. In this manner cDNA was generated from individual clonogenic OCI/AML-4 cells (sibling cells able to generate between 9-100 cells) or from individual non-clonogenic OCI/AML-4 cells (sibling cells were not able to generate more than 8 cells). Respective single cell cDNA was then pooled and compared by hybridization to cDNA microarrays. Using early passages of the OCI/AML-4 cell line cDNA was obtained from 17 clonogenic single cells (sibling cells able to generate between 9-100 cells) and 20 non-clonogenic single cells (sibling cells were not able to generate more than 8 cells). These cDNAs were pooled and hybridized to the cDNA microarray was conducted as three replicates, including one replicate which was a dye-swap.

由于难以分离获得具备明确增殖能力的纯细胞群体，调控急性髓系白血病（AML）细胞克隆形成能力（clonogenicity）的基因鉴定工作长期受阻。本研究以早期传代的OCI/AML-4细胞系为研究对象，旨在鉴定克隆形成细胞与非克隆形成细胞之间的差异表达基因。此前我们已证实，OCI/AML-4细胞的克隆源性同胞姊妹细胞呈现协同生长模式，因此可通过其克隆源性同胞姊妹细胞的生长模式，预测在制备全局单细胞互补脱氧核糖核酸（cDNA）时被裂解的单个细胞的增殖行为。我们将细胞以有限稀释法接种于96孔板中，随后筛选出由4个细胞组成的局部细胞簇，并通过显微操作将其中3个细胞分别转移至含有生长培养基及OP9细胞饲养层（feeder layer）的独立微量滴定孔内，同时将第4个细胞裂解后进行全局逆转录聚合酶链式反应（RT-PCR）处理。每2-3天对每个培养孔内的细胞进行计数，直至细胞停止生长。通过该方法，我们分别从单个克隆形成性OCI/AML-4细胞（即可增殖至9-100个细胞的同胞姊妹细胞）以及单个非克隆形成性OCI/AML-4细胞（即增殖能力不超过8个细胞的同胞姊妹细胞）中制备得到cDNA。随后将各组单细胞cDNA分别混合，通过与cDNA微阵列（cDNA microarray）杂交进行表达差异比较。本次研究采用早期传代的OCI/AML-4细胞系，最终从17株克隆形成性单细胞（可增殖至9-100个细胞的同胞姊妹细胞）及20株非克隆形成性单细胞（增殖能力不超过8个细胞的同胞姊妹细胞）中成功获取cDNA。将上述cDNA混合后与cDNA微阵列进行杂交，实验共设置三次生物学重复，其中一次重复采用染料交换（dye-swap）方案。