1 State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy, Nankai University , Tianjin, China .
2 Reproductive Medical Center, Department of Gynecology and Obstetrics, Peking University Third Hospital , Beijing, China .
Stem Cells Dev. 2018 Apr 1;27(7):479-487. doi: 10.1089/scd.2017.0193.
Haploid cells facilitate genetic screening of recessive mutations for a single set of chromosomes. Haploid embryonic stem cells (haESCs) have been achieved in several species and widely utilized in genetic screens. The fact that haESCs undergo substantial diploidization during differentiation has limited the screening to other haploid cell types. In this study, we report a method to establish haploid neural stem cells (haNSCs) by selection for a Pax6 reporter. We inserted a green fluorescence protein (GFP) marker gene by homologous recombination into the Pax6 locus of an haESC line. GFP-positive haploid cells could be sorted and further cultured in the NSC medium for more than 30 passages. The established haNSCs expressed neural lineage markers and could differentiate into neurons, oligodendroglia, and astrocytes. Our study shows the feasibility of deriving haploid proliferative somatic cell lines using a genetically encoded reporter that suggest a system for genetic screening of neural and retinal development.
单倍体细胞有利于对一组染色体的隐性突变进行遗传筛选。在几个物种中已经实现了单倍体胚胎干细胞（haESCs），并广泛应用于遗传筛选。事实上，haESCs 在分化过程中会经历大量的二倍体化，这限制了其他单倍体细胞类型的筛选。在这项研究中，我们报告了一种通过选择 Pax6 报告基因来建立单倍体神经干细胞（haNSCs）的方法。我们通过同源重组将绿色荧光蛋白（GFP）标记基因插入到 haESC 系的 Pax6 基因座中。GFP 阳性的单倍体细胞可以通过分选进一步在 NSC 培养基中培养超过 30 代。建立的 haNSCs 表达神经谱系标记物，并能分化为神经元、少突胶质细胞和星形胶质细胞。我们的研究表明，使用遗传编码报告基因来衍生单倍体增殖体细胞系是可行的，这为神经和视网膜发育的遗传筛选提供了一个系统。
