In Vitro Erythroid Differentiation and Lentiviral Knockdown in Human CD34+ Cells from Umbilical Cord Blood.
In Vitro Erythroid Differentiation and Lentiviral Knockdown in Human CD34+ Cells from Umbilical Cord Blood.
复制标题
DOI：
10.1007/978-1-4939-7428-3_16
复制
发表时间：
2018
影响因子：
--
通讯作者：
Anna Kovilakath;Safa F. Mohamad;F. Hermes;Shou-Zhen Wang;G. Ginder;J. Lloyd
中科院分区：
文献类型：
--
作者：
Anna Kovilakath;Safa F. Mohamad;F. Hermes;Shou-Zhen Wang;G. Ginder;J. Lloyd
人脐带血是造血干细胞和祖细胞的丰富来源。脐带血中的CD34+细胞比外周血或骨髓中的CD34+细胞更原始，并且可以高速增殖并分化为多种细胞类型。在该方案中，描述了一种可靠的方法，用于从脐带血中分离胎儿CD34+细胞并在培养中扩增这些细胞。然后，细胞可以在体外沿红系途径沿着分化，同时进行所选基因的敲低。使用表达小发夹RNA（shRNA）的慢病毒载体是下调基因的有效方法。流式细胞术分析用于富集红系细胞。使用这些方法，可以产生体外分化的细胞以用于定量逆转录酶PCR和其他目的。
Human umbilical cord blood is a rich source of hematopoietic stem and progenitor cells. CD34+ cells in umbilical cord blood are more primitive than those in peripheral blood or bone marrow, and can proliferate at a high rate and differentiate into multiple cell types. In this protocol, a dependable method is described for the isolation of fetal CD34+ cells from umbilical cord blood and expanding these cells in culture. The cells can then be in vitro differentiated along an erythroid pathway, while simultaneously performing knockdown of a gene of choice. The use of lentiviral vectors that express small hairpin RNA (shRNA) is an efficient method to downregulate genes. Flow cytometric analyses are used to enrich for erythroid cells. Using these methods, one can generate in vitro differentiated cells to use for quantitative reverse transcriptase PCR and other purposes.