Tulpule Asmin, Daley George Q
Harvard Medical School, Karp Family Research Building, Division of Hematology/Oncology, Children's Hospital Boston, Boston, MA, USA.
Methods Mol Biol. 2009;482:35-42. doi: 10.1007/978-1-59745-060-7_3.
Human embryonic stem cells (hESCs) represent a powerful platform to study human development and its dysfunction in human disease. However, certain biological properties have hampered the application of standard gain of function and loss of function tools to these cells. For example, while traditional gene knockouts by homologous recombination (HR) have been reported, the low cloning efficiency of hESCs has made HR a lengthy and laborious undertaking. An alternative method of achieving loss of function is the use of small interfering RNAs (siRNAs) that can be introduced either as pre-synthesized duplexed oligonucleotides or via lentiviral vector. The use of a lentiviral vector to deliver siRNAs has proven to be a rapid and specific way to achieve highly efficient and persistent gene knockdowns in hESCs. In this chapter, we will summarize the key requirements for the successful application of lentiviral RNAi in hESCs.
人类胚胎干细胞（hESCs）是研究人类发育及其在人类疾病中功能失调的强大平台。然而，某些生物学特性阻碍了标准功能获得和功能丧失工具在这些细胞中的应用。例如，虽然已有通过同源重组（HR）进行传统基因敲除的报道，但hESCs的低克隆效率使HR成为一项漫长而费力的工作。实现功能丧失的另一种方法是使用小干扰RNA（siRNAs），它们可以作为预先合成的双链寡核苷酸引入，也可以通过慢病毒载体引入。事实证明，使用慢病毒载体递送siRNAs是在hESCs中实现高效且持久的基因敲低的快速且特异的方法。在本章中，我们将总结在hESCs中成功应用慢病毒RNA干扰的关键要求。
