Atg7 Knockout Cells

Atg7, which is related to autophagy, is an essential protein for cell degradation and recycling. This sequence is related to the ubiquitin-proteasome system UPS required for the unique development of autophagosome membranes and intracellular fusion. During the initiation of autophagy, Atg7 acts like E-1 enzyme on ubiquitin-like proteins (UBL), such as Atg12 and Atg8. knocking cell line Atg7 to bind to these UBL proteins and activate its transfer to the E-2 enzyme to help its targeting molecules. Atg7's role in these two autophagy-specific UBL systems makes it an important regulator of autophagosome assembly.

Atg7 is required for acrosome biogenesis in mouse spermatogenesis

The acrosome is a special organelle that covers the front part of the sperm nucleus and plays a vital role in the fertilization process. The molecular mechanism of the biogenesis of this lysosomal-related organelle (LRO) is still unknown. In this study, mice with Atg7 knockout germ cells became sterile due to defective acrosome biogenesis and showed a phenotype similar to human coccidiosis. This reproductive defect can be successfully rescued by injecting sperm in the cytoplasm. In addition, knocking out Atg7 in germ cells does not affect the early stages of germ cell development, but in the later stages of spermatogenesis, precursor vesicles cannot fuse into a single acrosomal vesicle in the Golgi stage, resulting in irregular or almost Sperm with round head. The autophagy flux is destroyed in the germ cells of Atg7 gene knockout, which eventually leads to the failure of LC3 binding to Golgi-derived vesicles. In addition, in the process of acrosome biogenesis, Atg7 partially regulates another protein related to sperm disease, namely Golgi-related PDZ and a protein containing a coiled-coil motif (GOPC). Finally, injection of autophagy or lysosomal inhibitors into the testis produces a phenotype similar to germ cell-specific Atg7 knockout mice.

Germ cell-specific Atg7 gene knockout causes primary ovarian insufficiency in female mice

Among women under 40, about 1-2% of primary ovarian insufficiency (POI) is a common cause of infertility. However, the mechanism leading to POI is still poorly understood. In this case, germ cell-specific knockout of the necessary autophagy-inducing gene Atg7 resulted in infertility in female mice. The subfertility of Atg7 knockout women is caused by severe follicle loss, which is very similar to human POI patients. The mutation of Atg7 germ cells or the defect of autophagy mechanism may be the cause of POI. Further studies have shown that knockout of Atg7 gene specific to germ cells leads to excessive loss of germ cells in the transitional period of newborn. In addition, in vitro studies have also shown that autophagy can protect the oocytes of the newborn ovaries under starvation from excessive loss of apoptosis.

Similar to the POI of human patients, the ovarian follicle pool of Atg7 knockout mice is drastically reduced. Therefore, Atg7 mutation or defects in autophagy mechanism may be the cause of POI.

Autophagy defects in vascular smooth muscle cells promote cell death and atherosclerosis

Macroautophagy/autophagy is considered to be an evolutionarily conserved cell catabolic process. Here, the researchers studied the effect of autophagy on vascular smooth muscle (VSM) cells in atherosclerosis. VSM cells cultured in mice knocked out of the essential autophagy gene atg7 showed serum-induced reduction in cell growth, increased cell death and decreased cell proliferation rate. In addition, in VSM cells derived from Atg7 knockout mice, TRP53 (the mouse ortholog of human and rat TP53) was activated, and 7-ketocholesterol enhanced apoptosis and CCL2 (chemokine [ CC motif] ligand 2) expression. In addition, Atg7 knockout mice crossed with Apoe (apolipoprotein E) knockout mice showed reduced medial cell proliferation and increased TUNEL positive cells in the descending aorta at 10 weeks of age. Interestingly, the Atg7 knockout mice were crossed with Apoe knockout mice and fed a Western diet containing 1.25% cholesterol for 14 weeks, their survival rate decreased. Analysis of the descending aorta of Atg7 knockout mice crossed with Atoe7 knockout mice showed increased plaque area, increased TUNEL positive area, decreased VSM positive cell area, and accumulation of macrophages in the medium. Autophagy defects in VSM cells can enhance atherosclerotic changes through arterial outward remodeling.

CRISPR-U™ (based on CRISPR/Cas9 technology) developed by Ubigene is more efficient than ordinary CRISPR/Cas9 in double-strand breaks, and CRISPR-U™ can greatly improve the efficiency of homologous recombination and easily achieve knock-out (KO)) , Point mutation (PM) and knock-in (KI) in vitro and in vivo. With the help of CRISPR-U, Ubigene has successfully edited genes on more than 100 cell lines.

Reference

Wang H, Wan H, Li X, et al. Atg7 is required for acrosome biogenesis during spermatogenesis in mice. Cell Res. 2014;24(7):852-869. doi:10.1038/cr.2014.70 

Song ZH, Yu HY, Wang P, et al. Germ cell-specific Atg7 knockout results in primary ovarian insufficiency in female mice. Cell Death Dis. 2015;6(1):e1589. Published 2015 Jan 15. doi:10.1038/cddis.2014.559

Osonoi Y, Mita T, Azuma K, et al. Defective autophagy in vascular smooth muscle cells enhances cell death and atherosclerosis. Autophagy. 2018;14(11):1991-2006. doi:10.1080/15548627.2018.1501132

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.