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Invited researcher Prim Singh
Contract number
14.Y26.31.0024
Time span of the project
2018-2020

As of 15.02.2021

28
Number of staff members
14
scientific publications
General information

Name of the project: Non-medical heritage: conservative mechanisms of genetic imprinting

Strategy for Scientific and Technological Development Priority Level: в

Goals and objectives

Research directions: Mechanisms of genetic imprinting in animals

Project objective: Research of the role of conservative heterochromatin factors in regulation of imprinting in animals


The practical value of the study

  • The Laboratory has created colonies P. citri and S. coprophila at the Novosibirsk State University (Russia). P. citri lines have been taken from the University of Tuscany (Italy) and the University of Edinburgh (United Kingdom). At the Laboratory all the necessary conditions have been set up for creation and multiplication of P. citri, stable colonies have been produced that ensures sufficient amount of biological material for conducting experiments. S. coprophila lines have been transported from the University of Edinburgh (United Kingdom) and received from the Brown University (USA). Currently in the collection 7 lines of S. coprophila are supported that carry various genetic markers and chromosome abnormality necessary for work.
  • To produce fragments of genes of the H3K9 demethylase and genes of the Н3К9 and Н4К20 methylases we have conducted bioinformatical search in a draft assembly of the P. citri genome for homologs of corresponding genes in Drosophila melanogaster. As a result, for each gene of Drosophila melanogaster we have found from 1 to 5 homologs in the P. citri genome. Fragments of genes for further production of double-stranded RNAs was made using specific primers carrying sequences of promoter of T7 phage RNA polymerase on their 5' ends. P. citri genomic DNA or a sample of сDNA prepared from total RNA of adult females was used as a PCR-array. The produced fragments were copied into plasmid vectors and verified by sequencing. Similar work has been done for S. coprophila. As a result, we produced fragments of genes of the H3K9 demethylase and genes of the Н3К9 and the Н4К20 methylase from two model organisms that were prepared for production of samples of dsRNA for subsequent RNA interference.
  • From the Charité clinic (Berlin, Germany) we have imported mouse embryonic stem cells in which genes encoding HP1beta and HP1gamma are surrounded by LoxP sites. Encoding sequences of these genes can be removed using transient transfection with a construct expressing the Cre recombinase. According to the work plane we are expecting to use the effect of removal of all the HP1 paralogs on methylation of differentially methylated regions of DNA in imprinting control loci. Thus, before the beginning of the experiment we needed to remove the CBX5 gene that encodes the HP1alpha protein. It has been demonstrated earlier that removal of this protein does not affect livelihood of embryonic stem cells of mice. To remove the CBX5 gene we used a method that utilizes the CRISPR/Cas9 system. For this purpose we have prepared a guide RNAs whose recognition sites are located on the sides of the encoding part of the CBX5 gene and tested the RNAs for specificity. After their efficiency had been checked in a test system, we conducted Reconstruction of structures expressing these guide RNAs and the structure expressing the Cas9 nuclease. As a result of activity of Cas9 we expected to produce colonies of cells in which the encoding sequence of the CBX5 gene is removed in both homologs of chromosomes. After the transfection we picked 87 clones that we checked using PCR for existence of 13 t.b.p. deletion that leads to deletion of the encoding part of CBX5. IN the results of PCR we found 4 clones carrying the needed deletion on both homologs of chromosomes. Presence of the deletion was confirmed by sequencing. To eliminate the possibility of translocation of the CBX5 gene as a result of chromatin remodeling we measured expression of the CBX5 gene in the obtained clones using quantitative PCR. In all the 4 clones expression of the CBX5 gene was not detected.
  • Further work in this direction in related to detecting the level of methylation of DNA in loci of imprinting control using COBRA and bisulfite sequencing. To conduct research using these methods we selected primers for PCR that overlap regions of methylation of the 4 model loci selected for further work.

Education and career development: Internships at the Laboratory have been organized for 5 postgraduates and 8 students.

 Organizational and structural changes: An educational laboratory has been created for students of the Department of Cytology and Genetics and the Department of Molecular Biology of the Faculty of Natural Sciences of the Novosibirsk State University (Russia).

Collaborations:

  • University of Edinburgh (United Kingdom): harnesses methods of work with Plannococcus citri and Sciara coprophila model organisms, scientific internships
  • Institute of Cell Biology and Neurobiology at the Charité University Clinic (Germany): mastering methods of work with embryonic stem cells of mice, scientific internships
  • RIKEN (Japan): characterization of the epigenetic state and the level of expression of imprinted loci in mouse embryonic stem cells after removal of genes encoding proteins of the HP1 family
  • University of Tuscany (Italy): conducting experiments in RNA interference of genes encoding in embryos of Plannococcus citri
  • University of Antwerp (Belgium): new cell lines needed for further research have been produced via the method of genome engineering using CRISPR

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