LIFE SCIENCE

The Nankai University team found a “polishing” gene screening weapon


Since the advent of haploid embryonic stem cells, they have provided an excellent platform for forward genetics screening, and have been widely used in the screening of key regulatory genes such as various viral receptors, drug targets, and biological development phenomena. However, in the process of daily culture and differentiation, diploidization often occurs, which seriously affects its function of “gene screening weapon”. How to fundamentally inhibit diploidization and efficiently maintain the haploplography of haploid embryonic stem cells has become an important topic in the field of stem cell research.

In this regard, the research group of Shuai Ling, a researcher at the State Key Laboratory of Medicinal Chemical Biology of Nankai University, introduced the exogenous anti-apoptotic gene BCL2 into mouse haploid embryonic stem cells, and successfully constructed mouse haploid embryonic stem cell lines (haESCs) with BCL2 overexpression (BCL2-OE), which can maintain their haploavailability for a long time under harsh conditions such as in vitro and in vivo differentiation.

The results not only further confirm that anti-apoptosis can effectively inhibit the spontaneous diploidization of haploid cells, but also provide a solid foundation for promoting the target screening of haploid cells in the direction of drug screening and cell differentiation, and further “polish” the gene screening weapon of haploid cells. A few days ago, the paper introducing the results was published online in the well-known academic journal “Cell Proliferation” in the field of biology.

Schematic diagram of BCL2-OE effectively maintaining haploid properties in daily culture and in vivo and in vivo differentiation in vitro and in vivo The project team is pictured

According to reports, half of the genetic information of eukaryotes comes from the father, half from the mother, this “double backup” can resist adverse mutations in the genetic process, ensure species reproduction, but for genetic research, especially for the exploration of recessive genetic function is very limited. Haploid cells with only one set of chromosomes have no gene “backup”, and changes to any gene will be directly reflected in phenotypic changes. This is very beneficial for exploring life phenomena and cracking genetic codes.

However, tool cells of this class of artificial homozygotes tend to spontaneously diploid during daily culture or differentiation, thereby losing the advantage of a single-copy genome. Therefore, in daily culture, haploid needs to be continuously enriched and maintained by flow cytometry technology based on DNA content, which greatly limits the wider application of haploid cells. There is an urgent need to develop strategies to effectively inhibit haploid cell diploidy.

“Based on the accumulation and experience in the early stage, we found that the occurrence of apoptosis may be a core factor in the diploidization of haploid cells. Therefore, we envisage the introduction of the exogenous anti-apoptotic gene BCL2 in order to obtain cell lines that can efficiently maintain haplo. ”

In this study, the haESCs of BCL2-OE against apoptotic genes – BCL2-OE were comprehensively demonstrated in terms of anti-apoptosis, genomic stability and differentiation and development potential, and found that BCL2-OE can effectively improve the anti-apoptosis and haplobactericity maintenance ability of cells under a variety of harsh conditions, without affecting the proliferation and differentiation potential of embryonic stem cells.

On this basis, the researchers systematically compared the haploid maintenance ability of BCL2-OE and wild-type haploid embryonic stem cells in daily culture, embryoid differentiation, chimeric embryo and teratoma formation, and found that BCL2-OE can greatly improve its haploid maintenance ability compared with wild-type haploid, and for the first time achieve the detection of a high proportion of haploid cell contribution in E10.5 chimeric embryos. Later, based on the analysis of transcriptome sequencing, the researchers found that BCL2-OE effectively activated the expression of hyaluronic acid synthetase gene Has2, and improved the anti-apoptosis ability and haplomaintenance ability of cells through the miR26b-Has2-HA-Caspase regulatory pathway. Overexpression of Has2 in haploid embryonic stem cells is also highly effective in maintaining haplo.

“This work not only confirms that haploid embryonic stem cells can theoretically differentiate into any cell type in the form of haploids, but also comprehensively promotes the development of haploid screening systems for lineage-specific screening in a wider variety of cell types.” The handsome leader said. (Source: China Science News, Wu Junhui, Chen Bin)

Related paper information:https://doi.org/10.1111/cpr.13498



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