A new 3D genome living cell imaging tool, TALE.

On April 7, Cell Research published a research paper (cover story) by Liu Guanghui and Xu Tao of the Institute of Biophysics of the Chinese Academy of Sciences, as well as a research paper entitled "Visualization of Aging-Associated Chromatin Alterations with an Engineered TALE System" in collaboration with the Qu Jing Research Group of the Institute of Zoology of the Chinese Academy of Sciences.
the study developed a new 3D genome living cell imaging tool, TALE, and used the system to achieve accurate imaging of telomere shortening and chromatin structure changes associated with aging, such as changes in silk particle composition.
, the study found that a reduction in the number of copies of DNA from nuclear kernels could serve as a new molecular marker for human aging.
the above-mentioned scientific research results have laid the foundation for understanding the nature of human aging at the genetic and pergenital levels.
recent years, the discovery and widespread use of new nucleic acid binding proteins such as CRISPR/Cas9 and TALE have made it possible to accurately image specific sequences of the genome.
However, there are still some shortcomings in the current imaging system based on dCas9 and TALE: the main problem with the dCas9 system is that it involves a variety of proteins and RNA components, and the dCas9 protein molecular weight is large, so the system is difficult to express instantaneously in mammalian cells, while using the system to image cells Nuclear background noise is also high; AL TALE systems, despite their small protein molecular weight and direct binding of DNA, are currently published papers, especially in genomic imaging studies using human cells, which rarely use the "gold standard" of 3D fluorescence in-place hybridization (3D-FISH) to rigorously confirm the accuracy of TALE imaging.
In this study, researchers analyzed the results of repeated genome sequences such as traditional TALE-mediated telomeres, silk grains, and nucleic kernel DNA to find that the use of traditional TALE marker genome repetition sequences produces abnormal aggregate plaques in cells that in most cases are removed from the DNA target sequence on the genome, greatly limiting THELE's use in chromosomal 3D imaging.
team screened a series of "dissolved peptides" that improve protein solubility in cells and found that the fusion expression of Thioredoxin and THALE, known as TALE, specifically removes the aggregated plaques associated with THALE imaging, maximizing TALE's effectiveness in chromatin 3D imaging.
experimental results show that compared with CRISPR/dCas9 system, TTALE has the advantages of high imaging signal-to-noise ratio and easy operation, and can be widely used in embryonic stem cells, induced omnicient stem cells, adult stem cells (interstitial stem cells and neural stem cells), end-differentiated cells (neurons and blood vessel smooth muscle cells), tumor cells and egg cells and other human cell types.
In addition, using this tool, the researchers first realized the living cell markers of 28S cytosome DNA and revealed that 28S CYG DNA is mainly distributed in the outer edge of the nucleic kernel region, which will provide important clues and research tools for understanding the function of cystic DNA and kernels in multiple life processes.
human aging and many aging-related diseases are accompanied by chromosomal structure changes or abnormalities, so it is important to explore the dynamic changes of chromosomal structure to better understand the mechanisms of aging and aging diseases.
genome repetition sequence is a "dark matter" implied in the human genome, usually wrapped in dense isochromytes, and is considered a useless "junk sequence."
because of the limitations of research methods, in most existing genomic information analysis, these "junk sequences" are often subjectively "ignored".
known as telomeres is also a genome repeat sequence, and telomere shortening has become one of the few important molecular markers in the field of aging research.
, however, little is known about whether aging is accompanied by changes in the repetitive sequences of the broader genome.
Researchers first observed changes in genomic structures such as telomere shortening and isochromat disorders associated with cell aging in Petri dishes using the TALE system, and for the first time at the single-cell level in mice with telomerase deficiency.
, using the TALE imaging system, the team found that the reduction in the number of DNA copies of nuclear kernels may be one of the biological clocks of human aging.
The reduction in the number of dna copies of kerucleosome DNA associated with human aging is as significant as the shortening of telomeres, and can be easily detected from the outer blood of older people, so it can be used as a new molecular marker to evaluate the process of human aging.
research provides a powerful research tool to reveal the three-dimensional structure of human chromatin and its dynamic changes in aging and disease.
addition, the establishment of new molecular markers of human aging will also contribute to the foundation and transformation of aging research.
The work was selected as a cover article by Cell Research, and at the same time, Brian K. Kennedy, director of the Buck Institute for Aging in the United States, published Research Highlight, which spoke highly of the work: (1) the successful realization of live cell imaging of nuclear kerucosome DNA is a landmark effort.
As an important cause of genomic instability, the reduction in the number of DNA copies of nuclear glycosomes may be one of the driving forces behind the aging of the cereligical organisms ;(, and the application of the TTALE system may not be limited to the study of physiological processes such as human aging, but will also have potential value for the study of human diseases such as autism and cancer, and even gene therapy.
" the work was carried out in collaboration with the Institute of Biophysics, the Animal Institute, the Third Hospital of Peking University, the Beijing Hospital of the National Health and Planning Commission, the Beijing Century Temple Hospital affiliated with the Capital Medical University, and Hangzhou Normal University.
, Xu Tao and Qu Jing, researchers at the Institute of Biophysics in Shanghai, are co-authors.
, an associate researcher at the Institute of Biophysics, Deng Liping, a doctoral student, and Xue Yanhong, a senior engineer, are the first authors.
this work by the Ministry of Science and Technology, the Natural Science Foundation and the Chinese Academy of Sciences and other projects, the relevant technology has applied for invention patents.
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