`Yuri Voziyanov. Genome Engineering. Flp/FRT and Cre/loxP site-specific DNA recombination
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The Jackson Laboratory



Research Tools


Feng Li
Genome engineering
Evolution of variants of Flp recombinase

Riddhi Shah
Genome engineering
Evolution of variants of Flp and Flp-like recombinases

Yue Li
Genome engineering

Eugenia Voziyanova
Evolution of variants of Flp recombinase
Cell replacement

Yuri Voziyanov
Associate Professor of Biology

Louisiana Tech University
School of Biological Sciences / Institute for Micromanufacturing
CTH 121
1 Adams Blvd.
Ruston, LA 71272

318.257.2694
voziyan@latech.edu

1991 - BS/MS, Kiev State University, Kiev, Ukraine
1996 - Ph.D., Institute of Molecular Biology and Genetics, Kiev, Ukraine
1997 - 2003 - Postdoctoral training, University of Texas at Austin and European Molecular Biology Laboratory, Heidelberg, Germany



RESEARCH

There are two main directions of our current research: advanced genome engineering using tailor-made site-specific DNA recombinases and cell replacement in tissues by genetically modified stem cells.


Genome engineering

Main goal of this research direction is to develop safe and efficient tools to manipulate mammalian genomes using variants of site-specific recombinases evolved to recognize pre-existed, target-like genomic sequences. These genomic sequences resemble native recombination targets for wild-type recombinases. Target-like genomic sequences can be found in a mammalian genome quite often: every 5-10 kb. To evolve recombinase variants specific for genomic target-like sequences, we use target-linked molecular evolution approaches: site-directed and random mutagenesis and DNA shuffling. In our research we mainly use yeast tyrosine site-specific recombinases: Flp, TD1, R1 and others.


Depending on the relative location and orientation of their targets (shown as red triangles), site-specific DNA recombinases can execute a variety of genome manipulation reactions.


Examples of genomic target-like sequences that resemble native recombination target for Flp recombinase, FRT (A),: in the human interleukin-10 gene (B) and LTRs of HIV-1 (C). These FRT-like sequences were identified using the TargetFinder program. The positions within the FRT-like-‘binding elements’ that match those in FRT (A) are denoted by upper case green letters. Red lower case letters indicate mismatch. TargetFinder scores for the FRT-like sites, normalized to a score of 100 for FRT, are shown at the right.




Model gene targeting in E. coli using Flp variant FV7, which was evolved to recombine FRT-like sequence FL-IL10A in the human IL10 gene. (A) schematics of the assay, (B) image of a plate after the targeting, (C) control digestion of the plasmid DNA isolated from white (w) or blue (b) colonies.




Testing FV7 in mammalian cells (CHO). Deletion of the EGFP gene flanked by FRT and FRT-like DNA sequence from the human IL10 gene (FL-IL10A) by FV7 leads to expression of DsRed gene (the reaction is diagrammed at the top). Tests were performed in CHO cells transiently transfected with the reporter plasmid (A) and in CHO cells, which have the reporter integrated into genome (B). 48 hours after transfection residual fluorescence of EGFP is seen in the cells, in which recombination took place; this reflects either incomplete recombination (in A) or long half-life of EGFP in CHO cells (A, B). After cells were split and colonies formed, no EGFP fluorescence was detected in the cells expressing DsRed.



Cell replacement

New direction of our research is replacement of old/senescent/dysfunctional cells in tissues using adult stem cells. We are interested in developing approaches to engineer adult stem cells to express certain genes with therapeutic properties, to replace certain genes with their allelic variants known to extend the life span or to prevent early onset of aging disorders, and to repair genetic defects in the stem cells by replacing defective genomic DNA with the ‘wild type’ one. These genetically engineered stem cells could then be used to replace cells in adult tissues. Currently we are working on replacing ‘old’ melanocytes in the hair bulbs of albino mice with the ‘new’ ones, in which the ability to produce melanin is restored.


A C57BL/6J mouse treated with c-kit-blocking antibodies ACK45 to deplete the amplifying populations of melanoblasts after hair depilation, which initiated the hair cycle. Since melanocytes in the hair bulbs in the treated areas are depleted, the hair that grows after depilation is white.



[Publications (PubMed database)]


Selected publications


  • Voziyanova E, Malchin N, Anderson RP, Yagil E, Kolot M, Voziyanov Y.
    Efficient Flp-Int HK022 dual RMCE in mammalian cells.
    Nucleic Acids Res., 2013

  • Anderson R.P., Voziyanova E., Voziyanov Y.
    Flp and Cre expressed from Flp-2A-Cre and Flp-IRES-Cre transcription units mediate the highest level of dual recombinase-mediated cassette exchange.
    Nucleic Acids Res., 2012

  • Shultz J.L., Voziyanova E., Konieczka J.H., Voziyanov Y.
    A genome-wide analysis of FRT-like sequences in the human genome.
    PLoS One, 2011

  • Malchin N, Molotsky T, Borovok I, Voziyanov Y, Kotlyar A.B., Yagil E, Kolot M.
    High efficiency of a sequential recombinase-mediated cassette exchange reaction in Escherichia coli.
    J. Mol. Microbiol. Biotechnol., 2010