ACE2 knockout cell line

ACE2 Angiotensin Converting Enzyme 2, an angiotensin converting enzyme (ACE-like exonuclease of the dipeptide carboxyl carboxypeptidase family), is mainly expressed in the vascular endothelial cells of the heart, kidney and testis. Carboxypeptidase angiotensin-converting enzyme 2 (ACE2) hydrolyzes the octapeptide angiotensin (Ang)-II to heptapeptide Ang-(1-7), which has an important protective effect on blood sugar. ACE2 knockout mice respond to glucose with impaired glucose tolerance and impaired insulin secretion. On the contrary, providing ACE2 gene therapy to the pancreas can improve the blood sugar and primarycell function of db/db mice, which is a common genetic model of obesity-induced diabetes.

RNA transfection of TALEN produces distant Ace2 knockout mice, showing that colitis is reminiscent of pathophysiology and inflammation

Angiotensin I converting enzyme 2 (ACE2) is a key factor in maintaining intestinal homeostasis. Dysregulation of homeostasis can cause inflammation of the colon (colitis), which can lead to life-threatening weakness and even cancer. Animal models are valuable alternatives in deciphering the pathology behind such human diseases and screening hypothetical therapeutic targets or paradigms. However, the development of disease models may be time-consuming and require technology, which may affect its application value. In this study, we directly injected in vitro transcribed mRNA encoding transcriptional activator-like effector nucleases (TALENs) into the cytoplasm of outbred Kunming mouse fertilized eggs, thereby genetically destroying the mouse Ace2 gene. Therefore, the efficiency of inducing somatic mutations is 57%, of which 39% are frameshift mutations. Moreover, all modifications are stably transferred during germline transmission. In Ace2 knockout cell line male mice (Ace2-/y), the researchers observed severe chemically induced colitis, which was characterized by weight loss, diarrhea, and shortened colon length. Histologically, Ace2 mutation causes leukocyte \\\\\\\\\\\\\infiltration and significant destruction of the intestinal mucosal barrier. In addition, the researchers found that the expression of inflammatory cytokines in the colon tissue of Ace2- /y mice increased. Overall, the data show that by zygotic injection of TALEN mRNA, high targeting efficiency and heritability can be achieved in a hybrid mouse model. In addition, the resulting Ace2-//y mice exhibited phenotypic characteristics reminiscent of colitis, and the researchers expect that such mice may be valuable in the study of gut microbiome or fecal transplantation.

Expression and function of ACE2/Angiotensin(1-7)/Ma axis in U-2 OS and MNNG-HOS in osteosarcoma cells

The renin-angiotensin system (RAS) interacts with the proliferation and proliferation of various types of solid tumors through its classic angiotensin converting enzyme (ACE)/angiotensin II/angiotensin II type 1 receptor (AT1R) axis Transfer related. In a mouse model of osteosarcoma, AT1R blockers can reduce tumor volume and reduce liver and lung metastasis. The expression and function of the alternative ACE2/Ang(1-7)/Ma axis in osteosarcoma remains to be studied. In this study, the basic components of the replacement RAS axis and the expression of interleukin (IL)-1β stimulation were analyzed, and the effect of Mas on the proliferation and/or migration of U-2 OS and MNNG-HOS osteosarcoma cells was studied. Quantitative polymerase chain reaction showed that these two cell lines all expressed Ang (1-7) peptidase ACE2, neutral endopeptidase 24.11, prolyl-endopeptidase, and Ang (1-7). Body Mas. IL-1β induces the expression of Mas mRNA and protein, which is related to the reduction of proliferation and migration. In contrast, small interfering RNA-mediated knockdown of Mas expression leads to increased cell proliferation. In conclusion, osteosarcoma cells express a functionally active alternative to the ACE2/Ang(1-7)/Mas axis. By reducing growth and preventing cancer metastasis, the induction and enhancement of this axis may be beneficial for the treatment of osteosarcoma. These effects can be achieved by direct administration of Mas agonists or indirectly by blocking the classic AngII RAS axis through ACE inhibitors or AT1R antagonists.

Angiotensin II regulates ACE and ACE2 in neurons through p38 mitogen-activated protein kinase and extracellular signal-regulated kinase 1/2 signaling

Brain ANG II plays an important role in modulating sympathetic function and homeostasis. The generation and degradation of ANG II are carried out, to a large extent, through the angiotensin-converting enzyme (ACE) and ACE2, respectively. In disease states, such as hypertension and chronic heart failure, central expression of ACE is upregulated and ACE2 is decreased in central sympathoregulatory neurons. In this study, researcher determined the expression of ACE and ACE2 in response to ANG II in a neuronal cell culture and the subsequent signaling mechanism(s) involved. A mouse catecholaminergic neuronal cell line (CATH.a) was treated with ANG II (30, 100, and 300 nM) for 24 h, and protein expression was determined by Western blot analysis. ANG II induced a significant dose-dependent increase in ACE and decrease in ACE2 mRNA and protein expression in CATH.a neurons. This effect was abolished by pretreatment of the cells with the p38 MAPK inhibitor SB-203580 (10 μM) 30 min before administration of ANG II or the ERK1/2 inhibitor U-0126 (10 μM). These data suggest that ANG II increases ACE and attenuates ACE2 expression in neurons via the ANG II type 1 receptor, p38 MAPK, and ERK1/2 signaling pathways.

CRISPR-U™ (based on CRISPR/Cas9 technology), developed by Ubigene, is more efficient than general CRISPR/Cas9 in double-strand breaking, and CRISPR-U™ can greatly improve the efficiency of homologous recombination, easily achieve knockout (KO), point mutation (PM) and knockin (KI) in vitro and in vivo. With CRISPR-U, Ubigene has successfully edit genes on more than 100 cell lines.

Reference

Liu, C., Xiao, L., Li, F. et al. Generation of outbred Ace2 knockout mice by RNA transfection of TALENs displaying colitis reminiscent pathophysiology and inflammation. Transgenic Res 24, 433–446 (2015). 

Ender SA, Dallmer A, Lässig F, Lendeckel U, Wolke C. Expression and function of the ACE2/angiotensin(1-7)/Mas axis in osteosarcoma cell lines U-2 OS and MNNG-HOS. Mol Med Rep. 2014;10(2):804-810. doi:10.3892/mmr.2014.2266

Xiao L, Haack KK, Zucker IH. Angiotensin II regulates ACE and ACE2 in neurons through p38 mitogen-activated protein kinase and extracellular signal-regulated kinase 1/2 signaling. Am J Physiol Cell Physiol. 2013;304(11):C1073-C1079. doi:10.1152/ajpcell.00364.2012

The efficiency of gene knock-out and cleavage can not only give people the ability to generate protein radical profiles and establish regulatory records, but also has many advantages, making it a particularly attractive recombinant protein expression system. First, it is carboxylated on glutamic acid and sulfated on tyrosine. Second, the operation is simple, and the recombinant protein can be quickly produced through transient gene expression. Third, it can be used for stable recombinant protein production. Some researchers used gene cell knockout and cutting efficiency systems to generate gene-edited cell lines, targeted sequencing of GLUL genomic loci, produced stable EPO cell lines, and discovered the mechanism of stable expression of recombinant erythropoietin in humans .

According to customer needs, Yuanjing Biotechnology designs a stable gene transfer knockout program based on the target gene

Scheme 1: Small-segment gene knockout program, gRNA is set in the introns at both ends of exon 2, and the number of bases encoded by the knockout exon is not 3 times, and the knockout can cause frameshift.
Scheme 2: Frameshift gene knockout scheme, gRNA is set on the exon, the number of missing bases is not 3 times, and frameshift mutation can occur after knockout.
Scheme 3: Large-segment gene knockout scheme, knock out the coding sequence of the entire gene to achieve the effect of large-segment knockout.