Research - Institute of Genetics - Mammalian Cell Research Unit - Artificial Chromosome and Stem Cell Research Laboratory

Róbert KATONA
senior research associate

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Roberta FAJKA-BOJA senior research associate
Gyöngyi HOLLÓ laboratory assistant
Kinga KATONÁNÉ SZÉKELY SZŰCS laboratory assistant
Réka NAGY B.Sc student

ARTIFICIAL CHROMOSOME AND STEM CELL RESEARCH LABORATORY

Mammalian artificial chromosomes

Mammalian artificial chromosomes (MACs) are safe, stable, non-integrating vectors with a large transgene-carrying capacity. Previously, a method was discovered in our laboratory to produce stable, properly segregating, and non-integrating artificial chromosomes in various mammalian cell lines. These MACs are purported to be devoid of gene-coding sequences beyond rDNA. Natural equivalents of our artificial chromosomes are the heterochromatic small supernumerary marker chromosomes (sSMCs). sSMCs are present in 0.043% of the human population and most frequently originate from chromosome 15 (chr15). Stable heterochromatic sSMC can be inherited without phenotypic effects. Additionally, there is a report of 8 healthy members of a three-generation family carrying an sSMC derived from the short arm of an acrocentric chromosome. Recently, we reported the presence and detailed analysis of a stable sSMC in four healthy members of a three-generation family. It is of particular interest that this three-generation sSMC is a very stable entity: adding up the age of carrier family members the sum is >140 years. This fact provides convincing evidence that tetrasomy of the chr15p-q11.2b segment is harmless. The three-generation transmission demonstrates that such a chromosome can stably persist without apparent structural changes through generations. Furthermore, transgenic mice were produced with our artificial chromosome, and these mice were healthy and handed down the MACs into their offsprings through several generations. Recently, an artificial chromosome expression system, including a pre-engineered platform MAC with multiple acceptor sites (Platform ACE), was also developed. This Platform ACE is pre-engineered to contain multiple recombination acceptor attP sites for the ACE Integrase. In the Platform ACE system (Figure 1A), an ACE Integrase expression vector is co-transfected with an ACE-targeting vector (ATV) into a cell line harboring the Platform ACE (Figure 1B). The transiently expressed ACE Integrase then catalyses the integration of the targeting vector onto the Platform ACE. The ATV is a plasmid-based shuttle vector which conveys a gene(s) of interest onto the Platform ACE by means of targeted recombination between the recombination acceptor attP sites present on the Platform ACE and the recombination donor attB site of the ATV, catalyzed by the ACE Integrase. A promoterless antibiotic resistance gene downstream from the attB donor site allows for the selection of targeted integration events since it replaces the other antibiotic resistance gene already on the Platform ACE and acquires its promoter (Figure 1C.).



Figure 1. The schematics show the site-specific integration process by which the ACE chromosome is targeted with therapeutic transgenes. (A) The Platform ACE contains multiple attP recombination acceptor sites for the ACE integrase. The attP site is situated between an SV40 promoter and the puromycin resistance gene, which is driven by this promoter. A Platform ACE chromosome is also shown. The chromosome is counterstained by DAPI (blue). The green fluorescent staining demonstrates the presence of mouse major satellite sequences, which are major components of the ACE chromosome. Red fluorescent staining designates the attP sequence carrying sites suitable for the site-specific integration of transgenes. (B) The ATV vectors carry the attB recombination site for the ACE integrase. There is a promoterless neomycin resistance gene immediately after the attB site. The ACE integrase catalyzes the site-specific recombination between the attB and attP sites and integrates the ATV vector into the ACE chromosome. (C) The integration event disconnects the puromycin resistance gene from its promoter and replaces it with the promoterless neomycin resistance gene, which acquires the SV40 promoter. Targeted, transgene-carrying cell lines can be selected for G418 resistance. A transgene-loaded ACE chromosome is also present at the bottom of this figure. The green fluorescent staining demonstrates the presence of mouse major satellite sequences, which are major components of the ACE chromosome. Red fluorescent staining demonstrates the presence of the “loaded” transgene on the ACE chromosome. The chromosome is counterstained by DAPI (blue).


Combined mammalian artificial chromosome-stem cell therapy

The combination of ACE and stem cell technologies offers a new strategy for stem cell-based therapies the efficacy of which was confirmed and validated by using a mouse model of a devastating monogenic disease, galactocerebrosidase deficiency (Krabbe's disease). Therapeutic ACEs were generated by sequence-specific loading of galactocerebrosidase transgenes into a platform ACE, and stable, pluripotent mouse embryonic stem cell lines were established with these chromosomes. The transgenic stem cells were thoroughly characterized and used to produce chimeric mice on the mutant genetic background (Twitcher mice). The lifespan of these chimeras increased more than fourfold compared to control mice (Figure 2.) verifying the feasibility of the development of ACE-stem cell systems for the delivery of therapeutic genes in stem cells to treat genetic diseases and cancers and to produce cell types for cell replacement therapies.



Figure 2.


Future directions include:

  • We plan to modify various stem cell types with mammalian artificial chromosomes for basic research and gene therapy experiments.
  • We also conduct mouse model experiments with the combined artificial chromosome-stem cell therapy for X-linked SCID, cancer, and neuromuscular diseases.

Selected publications

Toth A, Fodor K, Praznovszky T, Tubak V, Udvardy A, Hadlaczky G, Katona Rl: Novel Method to Load Multiple Genes onto a Mammalian Artificial Chromosome. PLOS ONE 9:(1) p. e85565. (2014)

Czeh A, Schwartz A, Mandy F, Szoke Z, Koszegi B, Feher-Toth S, Nagyeri G, Jakso P, Katona RL, Kemeny A, Woth G, Lustyik G. Comparison and evaluation of seven different bench-top flow cytometers with a modified six-plexed mycotoxin kit. CYTOMETRY PART A 00A: 00-00. (2013)

Marton A, Vizler C, Kusz E, Temesfoi V, Szathmary Z, Nagy K, Szegletes Z, Varo G, Siklos L, Katona RL, Tubak V, Howard OM, Duda E, Minarovits J, Nagy K, Buzas K Melanoma cell-derived exosomes alter macrophage and dendritic cell functions in vitro. IMMUNOLOGY LETTERS 148:(1) pp. 34-38. (2012)

Szebeni GJ, Kriston-Pal E, Blazso P, Katona RL, Novak J, Szabo E, Czibula A, Fajka-Boja R, Hegyi B, Uher F, Krenacs L, Joo G, Monostori E Identification of galectin-1 as a critical factor in function of mouse mesenchymal stromal cell-mediated tumor promotion. PLOS ONE 7: (7) p. e41372. (2012)

Dominik Jańczewski, Jing Song, Erzsébet Csányi, Lóránd Kiss, Péter Blazsó, Róbert L. Katona, Mária A. Deli, Guillaume Gros, Xu Jian Wei, G. Julius Vancso. Organometallic polymeric carriers for a redox triggered release of molecular payloads, JOURNAL OF MATERIALS CHEMISTRY 22:(13) pp. 6429-6435. (2012)

Péter Blazsó, Ildikó Sinkó, Tünde Praznovszky, Gyula Hadlaczky, Róbert L. Katona. 3.6-kb mouse cyclin C promoter fragment is predominantly active in testis, ACTA BIOLOGICA HUNGARICA 63:(1) pp. 26-37. (2012)

Fabian G, Farago N, Feher LZ, Nagy LI, Kulin S, Kitajka K, Bito T, Tubak V, Katona RL, Tiszlavicz L, Puskas LG. High-Density Real-Time PCR-Based in Vivo Toxicogenomic Screen to Predict Organ-Specific Toxicity. Int J Mol Sci. 2011;12(9):6116-34. (2011)

Katona RL, Vanderbyl SL, Perez CF. Mammalian artificial chromosomes and clinical applications for genetic modification of stem cells: an overview. Methods Mol Biol. 738: 199-216. (2011) PubMed PMID: 21431729.

Sinko I, Katona RL. Transfer of Stem Cells Carrying Engineered Chromosomes with XY Clone Laser System. Methods Mol Biol. 738: 183-98. (2011) PubMed PMID: 21431728.

Blazso P, Sinko I, Katona RL. Engineered chromosomes in transgenics. Methods Mol Biol. 738: 161-81. (2011) PubMed PMID: 21431727.

Katona RL. Dendrimer mediated transfer of engineered chromosomes. Methods Mol Biol. 738: 151-60. (2011) PubMed PMID: 21431726.

Keller-Pinter A, Bottka S, Timar J, Kulka J, Katona R, Dux L, Deak F, Szilak L. Syndecan-4 promotes cytokinesis in a phosphorylation-dependent manner. Cell Mol Life Sci. 67: (11) 1881-94. (2010) PubMed PMID: 20229236.

Kovács-Sólyom F, Blaskó A, Fajka-Boja R, Katona RL, Végh L, Novák J, Szebeni GJ, Krenács L, Uher F, Tubak V, Kiss R, Monostori E. Mechanism of tumor cell-induced T-cell apoptosis mediated by galectin-1. Immunol Lett. 127(2): 108-18. (2010) PubMed PMID: 19874850.

Katona RL, Sinkó I, Holló G, Szucs KS, Praznovszky T, Kereso J, Csonka E, Fodor K, Cserpán I, Szakál B, Blazsó P, Udvardy A, Hadlaczky G. A combined artificial chromosome-stem cell therapy method in a model experiment aimed at the treatment of Krabbe's disease in the Twitcher mouse. Cell Mol Life Sci. 65(23): 3830-8. (2008) PubMed PMID: 18850314.

Katona RL, Loyer P, Roby SK, Lahti JM. Two isoforms of the human cyclin C gene are expressed differentially suggesting that they may have distinct functions. Acta Biol Hung. 58(1): 133-7. (2007) PubMed PMID: 17385550. Duncan, A. and Hadlaczky, Gy. Chromosomal engineering. Curr. Opin. Biotech. 18(5): 420-424. (2007)

Katona RL and Lahti JM. Cyclin C. AfCS-Nature Molecule Pages (2006) doi:10.1038/mp.a000720.0

Katona RL and Lahti JM. Cyclin Dependent Kinase 8 (CDK8). AfCS-Nature Molecule Pages (2006) doi:10.1038/mp.a003009.01

Loyer P, Trembley JH, Katona R, Kidd VJ, Lahti JM. Role of CDK/cyclin complexes in transcription and RNA splicing. Cell Signal. 17(9): 1033-51. (2005) Review. PubMed PMID: 15935619.

Katona RL, Cserpán I, Fátyol K, Csonka E, Hadlaczky G. Transgenic mice, carrying an expressed anti-HIV ribozyme in their genome, show no sign of phenotypic alterations. Acta Biol Hung. 56(1-2): 67-74. (2005) PubMed PMID: 15813215.

Lindenbaum, M., Perkins, E., Csonka, E., Greene, A., Fleming, E., Hadlaczky, Gy., MacDonald, N., Maxwell, A., Perez, C. and Ledebur, H. Jr. The ACE System: engineering artificial chromosomes to rapidly generate high-expressing cell lines for manufacture of recombinant proteins. Nucleic Acids Res 32(21): e172. (2004)

Monteith, D.P., Leung, J.D., Borowski, A.H., Co, D.O., Praznovski, T., Jiric, F.R., Hadlaczky, Gy. and Perez, C.F. (2003). Pronuclear microinjection of purified artificial chromosomes for generation of transgenic mice: Pick-and-Inject Technique. In Mammalian Artificial Chromosomes: Methods and Protocols. Eds.: Sgaramella, V. and Eridani, S. Methods in Mol. Biol. 240: 227-242.

Cserpán I, Katona R, Praznovszky T, Novák E, Rózsavölgyi M, Csonka E, Mórocz M, Fodor K, Hadlaczky G. The chAB4 and NF1-related long-range multisequence DNA families are contiguous in the centromeric heterochromatin of several human chromosomes. Nucleic Acids Res. 2002 Jul 1; 30(13):2899-905. PubMed PMID: 12087175; PubMed Central PMCID: PMC117038.

Hadlaczky, Gy. Satellite DNA-bases artificial chromosomes for use in gene therapy. Curr. Opin. Mol. Ther. 3(2): 125-132. (2001)

Csonka E, Cserpán I, Fodor K, Holló G, Katona R, Kereső J, Praznovszky T, Szakál B, Telenius A, deJong G, Udvardy A, Hadlaczky G. Novel generation of human satellite DNA-based artificial chromosomes in mammalian cells. J Cell Sci. 113: 3207-16. (2000) PubMed PMID: 10954419.

deJong, G., Telenius, A.H., Telenius, H., Perez, C.F., Drayer, J.I. and Hadlaczky, Gy. Mammalian artificial chromosome pilot facility: Large-scale isolation of functional satellite DNA-based artificial chromosomes. Cytometry 35: 129-133. (1999)

Telenius, H., Szeles, A., Kereső, J., Csonka, E., Praznovszky, T., Imreh, S., Maxwell, A., Perez, C.F., Drayer, J.I. and Hadlaczky Gy. Stability of a functional murine satellite DNA-based artificial chromosome across mammalian species. Chromosome Res. 7(1): 3-7. (1999)

Holló G, Kereső J, Praznovszky T, Cserpán I, Fodor K, Katona R, Csonka E, Fátyol K, Szeles A, Szalay AA, Hadlaczky G. Evidence for a megareplicon covering megabases of centromeric chromosome segments. Chromosome Res. 4(3): 240-7. (1996) PubMed PMID: 8793209.

Kereső J, Praznovszky T, Cserpán I, Fodor K, Katona R, Csonka E, Fátyol K, Holló G, Szeles A, Ross AR, Sumner AT, Szalay AA, Hadlaczky G. De novo chromosome formations by large-scale amplification of the centromeric region of mouse chromosomes. Chromosome Res. 4(3): 226-39. (1996) PubMed PMID: 8793208.

Katona R, Szeles A, Hadlaczky G. Mouse euchromatin specific "genome-painting" with a LINE probe: a rapid method for identification and mapping of human chromosomes in mouse-human microcell hybrids by two-color FISH. Hereditas. 124(2): 131-5. (1996) PubMed PMID: 8782433.

Hadlaczky, Gy., Praznovszky, T., Cserpán, I., Kereső, J., Péterfy, M., Kelemen, I., Atalay, E., Szeles, A., Szelei, J., Tubak, V. and Burg, K. Centromere formation in mouse cells cotransformed with human DNA and a dominant marker gene. Proc. Natl. Acad. Sci. U.S.A. 88: 8106-8110. (1991)

Praznovszky, T., Kereső, J., Tubak, V., Cserpán I., Fátyol, K. and Hadlaczky, Gy. De novo chromosome formation in rodent cells. Proc. Natl. Acad. Sci. U.S.A. 88: 11042-11046. (1991)