Hello everyone, today I'd like to share an article on PNAS. The corresponding author of this paper is Peter g. Schultz from Scripps Research Institute in the United States. Their research team can gain new understanding of the complex biochemical processes and mechanisms in living cells by developing chemical tools such as non natural amino acid insertion technology and studying the molecular processes in living cells.
Since 2001, Peter G Schultz group has developed the technology of genetic codon expansion, which can encode non natural amino acids (ncaas) and express them in proteins after the completion of codon transformation. After the development in recent years, this technology has successfully inserted non natural amino acids into the proteins of bacteria, yeast, mammalian cells, some multicellular organisms such as plants and animals. This technique is an effective tool for manipulating protein structure and function. In this paper, the authors report a method to stably encode ncaas in human blood stem cells (HSCs) and their differentiated progenies. Once these transformed stem cells are transplanted, they can produce a complete hematopoietic system encoding ncaas. The method developed in this paper can also be applied to other cell populations and provide an effective tool for the study of human protein in vitro and in vivo.
Hematopoietic system is responsible for producing many kinds of cells in the blood. It depends on the self-renewal and multi-directional differentiation ability of hematopoietic stem cells. Human hematopoietic system can be reconstructed by transplanting human hematopoietic stem cells into immunodeficient mice. Coding unnatural amino acids in such a system can produce the whole human hematopoietic system containing ncaas. Based on this recognition, we first developed a gene expression system based on EBV for stable, long-term and efficient expression of tRNA in human hematopoietic stem cells. Then HEK293T was used as a model cell to test the expression of tRNA / AARS and EGFP. With the good expression effect on the model, the author then expressed it on human primary HSCs cells, and confirmed that the expression system can express efficiently, and does not affect the cell differentiation. Next, the author tested in vivo, transferred the expression system into CD34 cells, and implanted it in mice to confirm that it can maintain its ability of coding ncaas and Multi Chain differentiation.
Finally, the author successfully inserted the non natural lysine protected by BOC into the primary hematopoietic stem cells GFP + / CD34 +, and transplanted it to mice, tested the content of various differentiated cells, and confirmed that 20% of HSCs transplanted into mice successfully encoded ncaas. Although this proportion is not high, the author believes that if a more suitable system is established in vivo, it may increase this proportion, and at present it is enough to be used in many cell level studies.
In a word, the author developed a stable and efficient expression system based on EBV, encoded ncaas in human primary hematopoietic stem cells, transplanted into mice and engineered the hematopoietic system. This technology can be used as an effective research tool and extended to other ncaas, including photocrosslinking agents, bioprobes and chemically modified amino acids with functions.
Author: ZYL
Article link: https://www.pnas.org/content/117/16/8845.long
Article quotation: doi: 10.1073/pnas.1914408117