title: ÐÐ»Ð¸ÑÐ½Ð¸Ðµ Ð½Ð¾ÐºÐ°ÑÑÐ° Ð³ÐµÐ½Ð° Psme1 Ð½Ð° ÑÐ°Ð½Ð½ÑÑ Ð´Ð¸ÑÑÐµÑÐµÐ½ÑÐ¸ÑÐ¾Ð²ÐºÑ ÑÐ¼Ð±ÑÐ¸Ð¾Ð½Ð°Ð»ÑÐ½ÑÑ ÑÑÐ²Ð¾Ð»Ð¾Ð²ÑÑ ÐºÐ»ÐµÑÐ¾Ðº Ð¼ÑÑÐ¸
title: Ð²ÑÐ¿ÑÑÐºÐ½Ð°Ñ ÐºÐ²Ð°Ð»Ð¸ÑÐ¸ÐºÐ°ÑÐ¸Ð¾Ð½Ð½Ð°Ñ ÑÐ°Ð±Ð¾ÑÐ° Ð±Ð°ÐºÐ°Ð»Ð°Ð²ÑÐ°
titleType: Subtitle
title: The effect of Psme1 knockout on early differentiation of mouse embryonic stem cells
Creators: name: ÐÐ°ÐºÑÐ¸Ð¼Ð¾Ð², ÐÐ»ÐµÐºÑÐ°Ð½Ð´Ñ
Publisher: name: Ð¡Ð°Ð½ÐºÑ-ÐÐµÑÐµÑÐ±ÑÑÐ³ÑÐºÐ¸Ð¹ Ð¿Ð¾Ð»Ð¸ÑÐµÑÐ½Ð¸ÑÐµÑÐºÐ¸Ð¹ ÑÐ½Ð¸Ð²ÐµÑÑÐ¸ÑÐµÑ ÐÐµÑÑÐ° ÐÐµÐ»Ð¸ÐºÐ¾Ð³Ð¾
Year: 2024
Subjects: ÑÐ¼Ð±ÑÐ¸Ð¾Ð½Ð°Ð»ÑÐ½ÑÐµ ÑÑÐ²Ð¾Ð»Ð¾Ð²ÑÐµ ÐºÐ»ÐµÑÐºÐ¸
ÑÐ±Ð¸ÐºÐ²Ð¸ÑÐ¸Ð½-Ð¿ÑÐ¾ÑÐµÐ°ÑÐ¾Ð¼Ð½Ð°Ñ ÑÐ¸ÑÑÐµÐ¼Ð°
Ð¿Ð»ÑÑÐ¸Ð¿Ð¾ÑÐµÐ½ÑÐ½Ð¾ÑÑÑ
ÑÐ°Ð½Ð½ÑÑ Ð´Ð¸ÑÑÐµÑÐµÐ½ÑÐ¸ÑÐ¾Ð²ÐºÐ°
stem cells
proteasome
pluripotency
early differentiation

description: ÐÐ°Ð½Ð½Ð°Ñ ÑÐ°Ð±Ð¾ÑÐ° Ð¿Ð¾ÑÐ²ÑÑÐµÐ½Ð° Ð¸Ð·ÑÑÐµÐ½Ð¸Ñ ÑÐ¾Ð»Ð¸ Ð¿ÑÐ¾ÑÐµÐ°ÑÐ¾Ð¼Ð½Ð¾Ð³Ð¾ ÑÐµÐ³ÑÐ»ÑÑÐ¾ÑÐ° PA28Î±Î² Ð² ÑÐ°Ð½Ð½ÐµÐ¹ Ð´Ð¸ÑÑÐµÑÐµÐ½ÑÐ¸ÑÐ¾Ð²ÐºÐµ ÑÐ¼Ð±ÑÐ¸Ð¾Ð½Ð°Ð»ÑÐ½ÑÑ ÑÑÐ²Ð¾Ð»Ð¾Ð²ÑÑ ÐºÐ»ÐµÑÐ¾Ðº (Ð­Ð¡Ð) Ð¼ÑÑÐ¸. ÐÐµÑÐ²ÑÐ¹ ÑÑÐ°Ð¿ ÑÐ°Ð±Ð¾ÑÑ Ð·Ð°ÐºÐ»ÑÑÐ°Ð»ÑÑ Ð² Ð²ÐµÑÐ¸ÑÐ¸ÐºÐ°ÑÐ¸Ð¸ Ð½Ð¾ÐºÐ°ÑÑÐ°, Ð¿Ð¾Ð»ÑÑÐµÐ½Ð½Ð¾Ð³Ð¾ ÑÐ°Ð½ÐµÐµ Ð² Ð»Ð°Ð±Ð¾ÑÐ°ÑÐ¾ÑÐ¸Ð¸ Ð¿ÑÑÐµÐ¼ ÑÐµÐ´Ð°ÐºÑÐ¸ÑÐ¾Ð²Ð°Ð½Ð¸Ñ Ð³ÐµÐ½Ð¾Ð¼Ð° Ñ Ð¿Ð¾Ð¼Ð¾ÑÑÑ ÑÐ¸ÑÑÐµÐ¼Ñ CRISPR/Cas9. ÐÐ»Ñ ÑÑÐ¾Ð³Ð¾ Ð¸Ð· ÐºÐ»ÐµÑÐ¾ÑÐ½ÑÑ Ð»Ð¸Ð½Ð¸Ð¹, Ð½Ðµ ÑÐºÑÐ¿ÑÐµÑÑÐ¸ÑÑÑÑÐ¸Ñ Ð¿ÑÐ¾Ð´ÑÐºÑ Ð³ÐµÐ½Ð° Psme1, Ð±ÐµÐ»Ð¾Ðº PA28Î±, Ð±ÑÐ»Ð° Ð²ÑÐ´ÐµÐ»ÐµÐ½Ð° Ð³ÐµÐ½Ð¾Ð¼Ð½Ð°Ñ ÐÐÐ, Ð°Ð¼Ð¿Ð»Ð¸ÑÐ¸ÑÐ¸ÑÐ¾Ð²Ð°Ð½Ñ ÑÑÐ°ÑÑÐºÐ¸ Ð³ÐµÐ½Ð° Psme1 Ð² Ð¾Ð±Ð»Ð°ÑÑÐ¸ Ð¿ÑÐ¾ÑÐ¾ÑÐ¿ÐµÐ¹ÑÐµÑÐ°, Ð¿Ð¾ÑÐ»ÐµÐ´Ð¾Ð²Ð°ÑÐµÐ»ÑÐ½Ð¾ÑÑÑ ÐºÐ¾ÑÐ¾ÑÑÑ Ð±ÑÐ»Ð° Ð¿ÑÐ¾Ð²ÐµÑÐµÐ½Ð° ÑÐµÐºÐ²ÐµÐ½Ð¸ÑÐ¾Ð²Ð°Ð½Ð¸ÐµÐ¼ Ð¿Ð¾ Ð¡ÑÐ½Ð³ÐµÑÑ. Ð¢Ð°Ðº Ð¶Ðµ Ð±ÑÐ»Ð¾ Ð¿ÑÐ¾Ð²ÐµÑÐµÐ½Ð¾ Ð¾ÑÑÑÑÑÑÐ²Ð¸Ðµ Ð¸Ð·Ð¼ÐµÐ½ÐµÐ½Ð¸Ð¹ Ð² Ð¿Ð¾ÑÐ»ÐµÐ´Ð¾Ð²Ð°ÑÐµÐ»ÑÐ½Ð¾ÑÑÑÑ, ÑÐ²Ð»ÑÑÑÐ¸Ð¼Ð¸ÑÑ Ð½Ð°Ð¸Ð±Ð¾Ð»ÐµÐµ Ð²ÐµÑÐ¾ÑÑÐ½ÑÐ¼Ð¸ Ð¼ÐµÑÑÐ°Ð¼Ð¸ Ð¿Ð¾Ð±Ð¾ÑÐ½Ð¾Ð³Ð¾ ÑÐµÐ´Ð°ÐºÑÐ¸ÑÐ¾Ð²Ð°Ð½Ð¸Ñ (off-target). ÐÑÐ¾ÑÐ¾Ð¹ ÑÑÐ°Ð¿ ÑÐ°Ð±Ð¾ÑÑ Ð²ÐºÐ»ÑÑÐ°Ð» ÑÐµÑÐ¸Ñ ÑÐºÑÐ¿ÐµÑÐ¸Ð¼ÐµÐ½ÑÐ¾Ð² Ñ Ð¸ÑÐ¿Ð¾Ð»ÑÐ·Ð¾Ð²Ð°Ð½Ð¸ÐµÐ¼ Ð¿Ð¾Ð»ÑÑÐµÐ½Ð½ÑÑ ÐºÐ»ÐµÑÐ¾ÑÐ½ÑÑ Ð»Ð¸Ð½Ð¸Ð¹ Ð­Ð¡Ð Ð¼ÑÑÐ¸ Ñ Ð½Ð¾ÐºÐ°ÑÑÐ¾Ð¼ Ð³ÐµÐ½Ð° Psme1, Ð² ÐºÐ¾ÑÐ¾ÑÑÑ Ð±ÑÐ» Ð¿ÑÐ¾Ð°Ð½Ð°Ð»Ð¸Ð·Ð¸ÑÐ¾Ð²Ð°Ð½ Ð¿Ð»ÑÑÐ¸Ð¿Ð¾ÑÐµÐ½ÑÐ½ÑÐ¹ ÑÑÐ°ÑÑÑ ÐºÐ»ÐµÑÐ¾Ðº Ñ Ð¿Ð¾Ð¼Ð¾ÑÑÑ ÑÐµÑÐ°ÑÐ¾Ð¼Ð½Ð¾Ð³Ð¾ ÑÐµÑÑÐ°; Ð¿ÑÐ¾Ð²ÐµÐ´ÐµÐ½Ð° Ð½Ð°Ð¿ÑÐ°Ð²Ð»ÐµÐ½Ð½Ð°Ñ Ð´Ð¸ÑÑÐµÑÐµÐ½ÑÐ¸ÑÐ¾Ð²ÐºÐ° in vitro; Ð¾ÑÑÑÐµÑÑÐ²Ð»ÐµÐ½ Ð°Ð½Ð°Ð»Ð¸Ð· ÐºÐ»ÐµÑÐ¾ÑÐ½Ð¾Ð¹ Ð¿ÑÐ¾Ð»Ð¸ÑÐµÑÐ°ÑÐ¸Ð¸ Ð¸ ÑÑÐ¾Ð²Ð½ÐµÐ¹ Ð°ÐºÑÐ¸Ð²Ð½ÑÑ ÑÐ¾ÑÐ¼ ÐºÐ¸ÑÐ»Ð¾ÑÐ¾Ð´Ð° (ÐÐ¤Ð) Ð¼ÐµÑÐ¾Ð´Ð¾Ð¼ Ð¿ÑÐ¾ÑÐ¾ÑÐ½Ð¾Ð¹ ÑÐ¸ÑÐ¾Ð¼ÐµÑÑÐ¸Ð¸; Ð¾ÑÐµÐ½ÐµÐ½Ð° ÑÐºÑÐ¿ÑÐµÑÑÐ¸Ñ ÑÐ¿ÐµÑÐ¸ÑÐ¸ÑÐµÑÐºÐ¸Ñ Ð³ÐµÐ½Ð¾Ð² Ð¿ÑÐ¸ Ð¿Ð¾Ð¼Ð¾ÑÐ¸ ÐºÐ¾Ð»Ð¸ÑÐµÑÑÐ²ÐµÐ½Ð½Ð¾Ð¹ ÐÐ¦Ð . ÐÑÑÐ¾Ð´Ñ Ð¸Ð· Ð¿Ð¾Ð»ÑÑÐµÐ½Ð½ÑÑ ÑÐµÐ·ÑÐ»ÑÑÐ°ÑÐ¾Ð² Ð±ÑÐ»Ð¾ ÑÐ´ÐµÐ»Ð°Ð½Ð¾ Ð·Ð°ÐºÐ»ÑÑÐµÐ½Ð¸Ðµ, ÑÑÐ¾ Ð­Ð¡Ð Ð¼ÑÑÐ¸ Ñ Ð½Ð¾ÐºÐ°ÑÑÐ¾Ð¼ Ð³ÐµÐ½Ð° Psme1 ÑÐ¾ÑÑÐ°Ð½ÑÑÑ ÑÑÐ°ÑÑÑ Ð¿Ð»ÑÑÐ¸Ð¿Ð¾ÑÐµÐ½ÑÐ½Ð¾ÑÑÐ¸ Ð¸ ÑÐ¿Ð¾ÑÐ¾Ð±Ð½Ð¾ÑÑÑ Ð´Ð¸ÑÑÐµÑÐµÐ½ÑÐ¸ÑÐ¾Ð²Ð°ÑÑÑÑ Ð²Ð¾ Ð²ÑÐµ ÑÑÐ¸ Ð·Ð°ÑÐ¾Ð´ÑÑÐµÐ²ÑÑ Ð½Ð°Ð¿ÑÐ°Ð²Ð»ÐµÐ½Ð¸Ñ in vivo. ÐÐ´Ð½Ð°ÐºÐ¾ Ð½Ð¾ÐºÐ°ÑÑ Ð³ÐµÐ½Ð° Î±-ÑÑÐ±ÑÐµÐ´Ð¸Ð½Ð¸ÑÑ ÑÐµÐ³ÑÐ»ÑÑÐ¾ÑÐ° PA28Î±Î² Ð¿ÑÐ¸Ð²Ð¾Ð´Ð¸Ñ Ðº ÑÐ½Ð¸Ð¶ÐµÐ½Ð¸Ñ ÑÐµÐ¼Ð¿Ð¾Ð² Ð¿ÑÐ¾Ð»Ð¸ÑÐµÑÐ°ÑÐ¸Ð¸ Ð¸ Ð¿Ð¾Ð²ÑÑÐµÐ½Ð¸Ñ ÑÑÐ¾Ð²Ð½ÐµÐ¹ ÐÐ¤Ð Ð² Ð­Ð¡Ð Ð¼ÑÑÐ¸. Ð­ÑÐ¸ Ð´Ð°Ð½Ð½ÑÐµ Ð³Ð¾Ð²Ð¾ÑÑÑ Ð¾ Ð²Ð°Ð¶Ð½Ð¾Ð¹ ÑÐ¾Ð»Ð¸ Ð¸ÑÑÐ»ÐµÐ´ÑÐµÐ¼Ð¾Ð³Ð¾ ÑÐµÐ³ÑÐ»ÑÑÐ¾ÑÐ° Ð² Ð´Ð¸ÑÑÐµÑÐµÐ½ÑÐ¸ÑÐ¾Ð²ÐºÐµ ÑÑÐ²Ð¾Ð»Ð¾Ð²ÑÑ ÐºÐ»ÐµÑÐ¾Ðº. ÐÐ¾Ð»ÑÑÐµÐ½Ð½ÑÐµ Ð² ÑÐ°Ð±Ð¾ÑÐµ Ð´Ð°Ð½Ð½ÑÐµ Ð¿Ð¾Ð·Ð²Ð¾Ð»ÑÑ Ð² Ð´Ð°Ð»ÑÐ½ÐµÐ¹ÑÐµÐ¼ ÑÐ°ÑÑÐ¸ÑÐ¸ÑÑ Ð¿Ð¾Ð½Ð¸Ð¼Ð°Ð½Ð¸Ðµ ÑÑÐ½Ð´Ð°Ð¼ÐµÐ½ÑÐ°Ð»ÑÐ½ÑÑ Ð¼ÐµÑÐ°Ð½Ð¸Ð·Ð¼Ð¾Ð² Ð´Ð¸ÑÑÐµÑÐµÐ½ÑÐ¸ÑÐ¾Ð²ÐºÐ¸ Ð­Ð¡Ð, ÑÑÐ¾ Ð¼Ð¾Ð¶ÐµÑ Ð±ÑÑÑ ÐºÑÐ¸ÑÐ¸ÑÐµÑÐºÐ¸ Ð²Ð°Ð¶Ð½Ð¾ Ð² ÑÐ°Ð·Ð²Ð¸ÑÐ¸Ð¸ ÑÐµÐ³ÐµÐ½ÐµÑÐ°ÑÐ¸Ð²Ð½Ð¾Ð¹ Ð¼ÐµÐ´Ð¸ÑÐ¸Ð½Ñ Ð¸ Ð² Ð±Ð¸Ð¾Ð¼ÐµÐ´Ð¸ÑÐ¸Ð½ÑÐºÐ¸Ñ Ð¸ÑÑÐ»ÐµÐ´Ð¾Ð²Ð°Ð½Ð¸ÑÑ Ð½Ð° Ð¼Ð¾Ð´ÐµÐ»ÑÑ ÑÐ°Ð·Ð»Ð¸ÑÐ½ÑÑ Ð±Ð¾Ð»ÐµÐ·Ð½ÐµÐ¹ ÑÐµÐ»Ð¾Ð²ÐµÐºÐ° Ð½Ð° Ð¾ÑÐ½Ð¾Ð²Ðµ Ð¿Ð»ÑÑÐ¸Ð¿Ð¾ÑÐµÐ½ÑÐ½ÑÑ ÑÑÐ²Ð¾Ð»Ð¾Ð²ÑÑ ÐºÐ»ÐµÑÐ¾Ðº.
description: The GQW is devoted to the study of the effect of the proteasome activator PA28Î±Î² on the early differentiation of mouse embryonic stem cells (ESCs). The first stage of the GQW was to validate the CRISPR/Cas9-mediated Psme1 knockout cell lines previously obtained in the laboratory. For this purpose, genomic DNA was extracted from treated cells and the region of Psme1 gene surrounding the gRNA target site was amplified by PCR and Sanger-sequenced. Regions where the gRNA off-target event was predicted, was also checked for any insertion or deletion (indels). The second stage of the GQW was to study the function of PA28 Î±Î² in ESCs differentiation using various approaches, including teratoma assay; cell differentiation in vitro; propidium iodide (PI) cell viability flow cytometry; flow cytometry of intracellular reactive oxygen species (ROS) with DCF staining; quantitative PCR (qPCR). Based on the results obtained in the GQW, we conclude that Psme1 knockout does not affect self-renewal and pluripotency in mouse ESCs. However, PA28Î±Î²-deficient ESC exhibits a decreased proliferation rate and is characterized by increased ROS level.Â  These data suggest an important role for PA28Î±Î² regulator in ESC differentiation and may be useful for understanding of the fundamental mechanisms of ESC differentiation and the further development of regenerative medicine and for research of various diseases based on model stem cells.