1. In Vitro Erythroid Differentiation and Lentiviral Knockdown in Human CD34+ Cells from Umbilical Cord Blood.
- Author
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Kovilakath A, Mohamad S, Hermes F, Wang SZ, Ginder GD, and Lloyd JA
- Subjects
- Antigens, CD34 metabolism, Cell Culture Techniques, Cell Separation, Erythroid Cells cytology, Erythroid Cells metabolism, Flow Cytometry, Gene Expression, HEK293 Cells, Humans, Transfection, Cell Differentiation genetics, Fetal Blood cytology, Gene Knockdown Techniques, Genetic Vectors genetics, Hematopoietic Stem Cells cytology, Hematopoietic Stem Cells metabolism, Lentivirus genetics
- Abstract
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.
- Published
- 2018
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