Lacroix Pépin Nicolas, Chapdelaine Pierre, Rodriguez Yoima, Tremblay Jacques-P, Fortier Michel A
Département d'Obstétrique et Gynécologie Faculté de Médecine, Université Laval et Axe de reproduction, santé de la mère et de l'enfant du Centre de Recherche, du CHU de Québec (CHUL), QC, Canada.
Département d'Obstétrique et Gynécologie Faculté de Médecine, Université Laval et Axe de reproduction, santé de la mère et de l'enfant du Centre de Recherche, du CHU de Québec (CHUL), QC, Canada Département de Médecine Moléculaire, Faculté de Médecine, Université Laval et Axe Neuroscience du Centre de Recherche du CHU de Québec (CHUL), Québec, QC, Canada GIV 4G2.
Mol Hum Reprod. 2014 Jul;20(7):650-63. doi: 10.1093/molehr/gau023. Epub 2014 Mar 27.
Prostaglandins (PGs) are important regulators of female reproductive function. The primary PGs produced in the endometrium are PGE2 and PGF2α. Relatively little is known about the biosynthetic pathways leading to the formation of PGF2α. We have described the role of aldo-ketoreductase (AKR)1B1 in increased PGF2α production by human endometrial cells following stimulation with interleukin-1β (IL-1β). However, alternate PGF synthases are expressed concurrently in endometrial cells. A definite proof of the role of AKR1B1 would require gene knockout; unfortunately, this gene has no direct equivalent in the mouse. Recently, an efficient genome-editing technology using RNA-guided DNase Cas9 and the clustered regularly interspaced short palindromic repeats (CRISPR) system has been developed. We have adapted this approach to knockout AKR1B1 gene expression in human endometrial cell lines. One clone (16-2) of stromal origin generated by the CRISPR/Cas9 system exhibited a complete loss of AKR1B1 protein and mRNA expression, whereas other clones presented with partial edition. The present report focuses on the characterization of clone 16-2 exhibiting deletion of 68 and 2 nucleotides, respectively, on each of the alleles. Cells from this clone lost their ability to produce PGF2α but maintained their original stromal cell (human endometrial stromal cells-2) phenotype including the capacity to decidualize in the presence of progesterone (medroxyprogesterone acetate) and 8-bromo-cAMP. Knockout cells also maintained their ability to increase PGE2 production in response to IL-1β. In summary, we demonstrate that the new genome editing CRISPR/Cas9 system can be used in human cells to generate stable knockout cell line models. Our results suggest that genome editing of human cell lines can be used to complement mouse KO models to validate the function of genes in differentiated tissues and cells. Our results also confirm that AKR1B1 is involved in the synthesis of PGF2α.
前列腺素（PGs）是女性生殖功能的重要调节因子。子宫内膜产生的主要前列腺素是PGE2和PGF2α。关于导致PGF2α形成的生物合成途径，人们了解相对较少。我们已经描述了醛酮还原酶（AKR）1B1在白细胞介素-1β（IL-1β）刺激后人子宫内膜细胞中PGF2α产量增加中的作用。然而，替代的PG合成酶在子宫内膜细胞中同时表达。要明确证明AKR1B1的作用需要基因敲除；不幸的是，该基因在小鼠中没有直接对应的基因。最近，一种使用RNA引导的DNA酶Cas9和成簇规律间隔短回文重复序列（CRISPR）系统的高效基因组编辑技术已经开发出来。我们采用这种方法在人子宫内膜细胞系中敲除AKR1B1基因表达。由CRISPR/Cas9系统产生的一个基质来源的克隆（16-2）表现出AKR1B1蛋白和mRNA表达完全缺失，而其他克隆呈现部分编辑。本报告重点关注分别在每个等位基因上缺失68和2个核苷酸的克隆16-2的特征。来自该克隆的细胞失去了产生PGF2α的能力，但保持了其原始的基质细胞（人子宫内膜基质细胞-2）表型，包括在孕酮（醋酸甲羟孕酮）和8-溴-cAMP存在下蜕膜化的能力。敲除细胞也保持了其响应IL-1β增加PGE2产生的能力。总之，我们证明新的基因组编辑CRISPR/Cas9系统可用于人类细胞以产生稳定的基因敲除细胞系模型。我们的结果表明，人类细胞系的基因组编辑可用于补充小鼠基因敲除模型，以验证分化组织和细胞中基因的功能。我们的结果还证实AKR1B1参与PGF2α的合成。
