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A novel method for genetic transformation of yeast cells using oligoelectrolyte polymeric nanoscale carriers
 
Yevhen Filyak1, Nataliya Finiuk1,2, Nataliya Mitina3, Oksana Bilyk1, Vladimir Titorenko4, Olesya Hrydzhuk1, Alexander Zaichenko3, and Rostyslav Stoika1
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Figure 5.  Transformants number ofPichia pastoris GS115 his4yeast when using different transformation methods. (Click to enlarge)




Branched polyethylenimine (PEI) might be considered the closest commercially available analog to the polymeric carrier developed here. PEI has been widely used for DNA delivery in the mammalian cells (52-55). The mechanisms of action of the PEI, as well as our developed polymer, might be the following: (i) condensing DNA, (ii) providing protection against potential nuclease degradation, and (iii) promoting association with the plasma membrane of a target cell to enhance entry into the cell. Following an uptake, DNA should be capable of escape from the endosome and be released into the cytoplasm to be capable of reaching the nucleus. We found that PEI exhibited significantly lower transformation efficiencies with yeast cells, compared with our oligoelectrolyte polymeric nanoscale carriers. Transformation of Saccharomyces cerevisiae yeast

Saccharomyces cerevisiae yeast is a unicellular eukaryotic organism most intensively used and studied in molecular and cell biology; in addition, it is widely used in biotechnology (6, 7, 16, 56). Although current transformation methodologies are enough sufficient for S. cerevisiae, we were interested to see if our oligoeletrolyte polymeric carriers could further enhance transformation efficiency.

Comparison of transformation efficiency using a circular episomal plasmid DNA (pYEp352 of 5.2 kb (containing URA3 gene as a selectable marker), Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, Ukraine) and yeast Saccharomyces cerevisiae BY4742 MATα his3Δ leu2Δ lysΔ ura3Δ cells were performed. Ura+ transformants of S. cerevisiae was selected on a solid minimal modified Burkholder medium without uracil with transformants selected based on uracil independence.

Our polymeric carrier based transformation approach yielded 17,100 colonies of yeast transformants per 1 µg of DNA, while the standard electroporation protocol resulted in 1000 colonies, and chemical LiAc-based transformation yielded 8300 colonies (Figure 6).




Figure 6.  Transformants number ofSaccharomyces cerevisiaeBY4742 yeast when using different transformation methods. (Click to enlarge)




Thus, there is no significant increase in the transformation efficiency when using our carrier with S. cerevisiae cells whose surface is well permeable to plasmid DNA. However, a distinct advantage in the transformation efficiency could be demonstrated when using yeast species such as Hansenula polymorpha and Pichia pastoris which have proven less emmenable to transformation in previous studies. These findings strongly suggest that the developed polymeric carrier possesses chemical characteristics necessary for efficient DNA delivery. We can hypothesize that such characteristics could include the hydrophobic core chain required for the improved penetration through the plasma membrane or possibly the positively charged polymer branches involved in binding DNA molecule, although this remains to be further investigated.

It is important to note that the polymeric carrier is highly efficiency for both transient and stable transformation when using either linearized or circular plasmid DNA. Moreover, there is no need for additional pretreatment steps to preparing competent cells or the requirement of special equipment for conducting the transformation. In addition, the developed method of yeast transformation is more convenient and rapid in use, and the proposed DNA carrier exhibits low toxicity and is not mutagenic (Supplementary Figures S3 and S4). This is unlike the widely used chemical-based LiAc method, in which costly geneticine antibiotic or other reagents are required for positive selection, and, thus, cells have to be incubated for long periods of time after transformation. Such prolonged cell incubation without selection makes experiments time consuming and decreases significantly the reproducibility of the results. A detailed comparison between the various transformation approaches can be found in Table 2.


Table 2.  Comparison of the DNA transformation methods (Click to enlarge)


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In conclusion, we have developed a novel method for efficient delivery of DNA into yeast cells using polymeric nanocomposites. We clearly demonstrated this method is more efficient and reproducible than other currently used methods for genetic transformation such as LiAc and electroporation. The developed method yielded a higher number of genetic transformants for the yeast species H. polymorpha and P. pastoris which are known to have a very low efficiency using current methods. Thus, our polymeric carrier represents the first nanoscale carrier for DNA delivery into the yeast cells, an approach that previously had only been applied for DNA delivery into the mammalian cells.

Acknowledgments

This work was partly supported by the grants from WUBMRC (Ukraine-USA), CRDF (USA), and F-46 project of the National Academy of Sciences of Ukraine, as well as by the project funded by the Ministry of Education and Science of Ukraine. The authors thank V. Nazarko, PhD, A. Polupanov, Y. Ryabec, PhD, I. Bohovych, and Y. Pynyaha, PhD (all from the Institute of Cell Biology, NAS of Ukraine) for their great help and fruitful discussions.

Competing interests

Authors declare no competing interests.

Correspondence
Address correspondence to Rostyslav Stoika, Department of Regulation of Cell Proliferation and Apoptosis, Institute of Cell Biology, NAS of Ukraine, Drahomanov St. 14/16, Lviv, Ukraine. E-mail: [email protected]

References
1.) Mager, W.H., and J. Winderickx. 2005. Yeast as a model for medical and medicinal research. Trends Pharmacol. Sci. 26:265-273.

2.) Armaleo, D., G.N. Ye, T.M. Klein, K.B. Shark, J.C. Sanford, and S.A. Johnston. 1990. Biolistic nuclear transformation of Saccharomyces cerevisiae and other fungi. Curr. Genet. 17:97-103.

3.) Barry, M.A., A.K. Rana, and H.A. Anderson. 2003. Gene gun Technologies: Applications for gene Therapy and Genetic Immunization. Gene and Cell Therapy 3:263-285.

4.) Becker, D.M., and L. Guarente. 1991. Highefficiency transformation of yeast by electroporation. Methods Enzymol. 194:182-187.

5.) Bartel, P.L., and S. Fields. 1995. Analyzing protein-protein interactions using two-hybrid system. Methods Enzymol. 254:241-263.

6.) Butow, R.A., R.M. Henke, J.V. Moran, S.M. Belcher, and P.S. Perlman. 1996. Transformation of Saccharomyces cerevisiae mitochondria using the biolistic gun. Methods Enzymol. 264:265-278.

7.) Agatep, R., R.D. Kirkpatrick, D.L. Parchaliuk, R.A. Woods, and R.D. Gietz. 1998. Transformation of Saccharomyces cerevisiae by the lithium acetate/single-stranded carrier DNA/polyethylene glycol (LiAc/ss-DNA/PEG) protocol. Technical Tips Online http://tto.trends.com 1:P01525.

8.) Kato, M., H. Iefuji, K. Miyake, and Y. Iimura. 1997. Transformation system for a wastewater treatment yeast, Hansenula fabianii J640: isolation of the orotidine-5′-phosphate decarboxylase gene (URA3) and uracil auxotrophic mutants. Appl. Microbiol. Biotechnol. 48:621-625.

9.) Gietz, R.D., and R.A. Woods. 2001. Genetic transformation of yeast. Biotechniques 30:816-820.

10.) Kawakami, S., S. Harashima, A. Kobayashi, and K. Fukui. 2006. Transformation of yeast using bioactive beads with surface-immobilized yeast artificial chromosomes. Methods Mol. Biol. 349:61-65.

11.) Beach, D., M. Piper, and P. Nurse. 1982. Construction of a Schizosaccharomyces pombe gene bank in a yeast bacterial shuttle vector and its use to isolate genes by complementation. Mol. Gen. Genet. 187:326-329.

12.) Das, S., and C.P. Hollenberg. 1982. A high-frequency transformation system for the yeast Kluyveromyces lactis. Curr. Genet. 6:123-128.

13.) Hinnen, A., J.B. Hicks, and G.R. Fink. 1978. Transformation of yeast. Proc. Natl. Acad. Sci. USA 75:1929-1933.

14.) Tikhomirova, L.P., R.N. Ikonomova, and E.N. Kuznetsova. 1986. Evidence for autonomous replication and stabilization of recombinant plasmids in the transformants of yeast Hansenula polymorpha. Curr. Genet. 10:741-747.

15.) Struhl, K., D.T. Stinchcomb, S. Scherer, and R.W. Davis. 1979. High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc. Natl. Acad. Sci. USA 76:1035-1039.

16.) Dohmen, R.J., A.W. Strasser, C.B. Honer, and C.P. Hollenberg. 1991. An efficient transformation procedure enabling long-term storage of competent cells of various yeast genera. Yeast 7:691-692.

17.) Brzobohatý, B., and L. Kovac. 1986. Factors enhancing genetic transformation of intact yeast cells modify cell wall porosity. J. Gen. Microbiol. 132:3089-3093.

18.) Foote, S., and C. Denny. 2002. Construction of YAC libraries with large inserts. Curr. Protoc. Hum. Genet. Unit 5.2 5.

19.) Duan, J., Y. Zhang, W. Chen, C. Shen, M. Liao, Y. Pan, J. Wang, X. Deng, and J. Zhao. 2009. Cationic polybutyl cyanoacrylate nanoparticles for DNA delivery. J. Biomed. Biotechnol. 2009:149254.

20.) Zhong, Q., D.M. Chinta, S. Pamujula, H. Wang, X. Yao, T.K. Mandal, and R.B. Luftig. 2010. Optimization of DNA delivery by three classes of hybrid nanoparticle/DNA complexes. J. Nanobiotechnology 8:6.

21.) Polu, A.R., and R. Kumar. 2011. Impedance Spectroscopy and FTIR Studies of PEG - Based Polymer Electrolytes. E-J. Chem. 8:347-353.

22.) Costanzo, M.C., and T.D. Fox. 1988. Transformation of yeast by agitation with glass beads. Genetics 120:667-670.

23.) O'Brien, J.A., and S.C. Lummis. 2011. Nano-biolistics: a method of biolistic transfection of cells and tissues using a gene gun with novel nanometer-sized projectiles. BMC Biotechnol. 11:66.

24.) Uchida, M., X.W. Li, P. Mertens, and H.O. Alpar. 2009. Transfection by particle bombardment: delivery of plasmid DNA into mammalian cells using gene gun. Biochim. Biophys. Acta 1790:754-764.

25.) Zlotnik, H., M.P. Fernandez, B. Bowers, and E. Cahib. 1984. Saccharomyces cerevisiace mannoproteins from an external cell wall layer that determines wall porosity. J. Bacteriol. 159:1018-1026.

26.) Reddy, A., and F. Maley. 1993. Dithiothreitol improves the efficiency of yeast transformation. Anal. Biochem. 208:211-212.

27.) Kimura, A., A. Arima, and K. Murata. 1981. Biofunctional change in yeast cell surface on treatment with Triton X-100. Agric. Biol. Chem. 45:2627-2629.

28.) Klein, T.M., R. Arentzen, P.A. Lewis, and F. McElligott. 1992. Transformation of microbes, plants by particle bombardment. Biotechnology (N. Y.) 10:286-291.

29.) Ito, H., K. Murata, and A. Kimura. 1983. Transformation of yeast cells treated with 2-mercaptoethanol. Agric. Biol. Chem. 47:1691-1692.

30.) Ito, H., Y. Fukuda, K. Murata, and A. Kimura. 1983. Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153:163-168.

31.) Schiestl, R.H., P. Manivasakam, R.A. Woods, and R.D. Gietz. 1993. Introducing DNA into yeast by transformation. Methods 5:79-85.

32.) Hill, J., K.A. Donald, and D.E. Griffiths. 1991. DMSO-enhanced whole cell yeast transformation. Nucleic Acids Res. 19:5791.

33.) Woods, R.A., and R.D. Gietz. 2000.Yeast transformation. In P.A. Norton, and L.F. Steel (Eds.) Gene Transfer Methods: Introducing DNA into Living Cells and Organisms. Eaton Publishing, Natick, MA.

34.) Gietz, R.D., and R.A. Woods. 2002. Transformation of yeast by lithium acetate/single-stranded carrierDNA/polyethylene glycol method. Methods Enzymol. 350:87-96.

35.) Klebe, R.J., J.V. Harriss, Z.D. Sharp, and M.G. Douglas. 1983. A general method for polyethylene-glycol-induced genetic transformation of bacteria and yeast. Gene 25:333-341.

36.) Chen, D.C., B.C. Yang, and T.T. Kuo. 1992. One-step transformation of yeast in stationary phase. Curr. Genet. 21:83-84.

37.) Zaichenko, A.S., S.A. Voronov, O.M. Shevchuk, V.P. Vasilyev, and A.I. Kuzayev. 1998. Kinetic features and molecular weight characteristics of terpolymerization products of the systems based on vinyl acetate and 5-tert-butyl-peroxy-5-methyl -1-hexene-3-yne. J. Appl. Polym. Sci. 67:1061-1066.

38.) Zaichenko, A., N. Mitina, M. Kovbuz, I. Artym, and S. Voronov. 2001. Low-temperature surface-active complex-radical oligo (di-tert.alkyl) peroxide initiators and curing agents. Wiley-VCH 164:47-71.

39.) Zaichenko, A., N. Mitina, M. Kovbuz, I. Artym, and S. Voronov. 2000. Surface-active metal-coordinated Oligoperoxidic Radical Initiators. J. Polym. Sci. A Polym. Chem. 38:516-527.

40.) Shavlovsky, G.M., V.P. Zharova, I.F. Shchelokova, V.M. Trach, A.A. Sibirny, and G.P. Ksheminskaya. 1978. Flavinogenic activity of natural strains of the yeast Pichia guilliermondii. Prikl. Biokhim. Mikrobiol. in Russian 14:184-189.

41.) Sambrook, J., and D.W. Russell. 2001. Molecular cloning, a laboratory manual, Cold Spring Harbor Laboratory, 3 e. Cold Spring Harbor, N.Y.

42.) Dmytruk, K.V., O.V. Smutok, O.B. Ryabova, G.Z. Gayda, V.A. Sibirny, W. Schuhmann, M.V. Gonchar, and A.A. Sibirny. 2007. Isolation and characterization of mutated alcohol oxidases from the yeast Hansenula polymorpha with decreased affinity toward substrates and their use as selective elements of an amperometric biosensor. BMC Biotechnol. 7:33.

43.) Faber, K.N., G.J. Swaving, F. Faber, G. Ab, W. Harder, M. Veenhuis, and P. Haima. 1992. Chromosomal targeting of replicating plasmids in the yeast Hansenula polymorpha. J. Gen. Microbiol. 138:2405-2416.

44.) Smutok, O., K. Dmytruk, M. Gonchar, A. Sibirny, and W. Schuhmann. 2007. Permeabilized cells of flavocytochrome b2 over-producing recombinant yeast Hansenula polymorpha as biological recognition element in amperometric lactate biosensors. Biosens. Bioelectron. 23:599-605.

45.) Gellissen, G. 2002. Hansenula polymorpha: biology and applications. .

46.) Buckholz, R.G., and M.A. Gleeson. 1991. Yeast systems for the commercial production of heterologous proteins. Biotechnology (N. Y.) 9:1067-1072.

47.) Scharstuhl, A., H. Glansbeek, E.L. Vitters, P.M. Van der Kraan, and W.B. Van den Berg. 2003. Large scaleprotein production of the extracellular domain of the transforming growthfactor-type II receptor using the Pichia pastoris expression system. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 786:271-277.

48.) Razanov, D.V., and A.Y. Strongin. 2003. Membrane type-1 matrix metalloproteinase functions as a proproteinself-convertase. J. Biol. Chem. 278:8257-8260.

49.) Wang, Y., Z.H. Liang, Y.S. Zhang, S.Y. Yao, Y.G. Hu, Y.H. Tang, S.Q. Zhu, D.F. Cui, and Y.M. Feng. 2001. Human insulin from a precursor overexpression in the methylortophic yeast Pichia pastors and a simple procedure for purifying the expression product. Biotechnol. Bioeng. 73:74-79.

50.) Daly, R., and M.T. Hearn. 2005. Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production. J. Mol. Recognit. 18:119-138.

51.) De Schutter, K., Y.-C. Lin, and P. Tiels. 2009. Genome sequence of the recombinant protein production host Pichia pastoris. Nat. Biotechnol. 27:561-566.

52.) Ahn, H.H., J.H. Lee, K.S. Kim, J.Y. Lee, M.S. Kim, G. Khang, I.W. Lee, and H.B. Lee. 2008. Polyethyleneimine-mediated gene delivery into human adipose derived stem cells. Biomaterials 29:2415-2422.

53.) Lee, J.H., H.H. Ahn, K.S. Kim, J.Y. Lee, M.S. Kim, B. Lee, G. Khang, and H.B. Lee. 2008. Polyethyleneimine-mediated gene delivery into rat pheochromocytoma PC-12 cells. J. Tissue Eng. Regen. Med. 2:288-295.

54.) Xia, T., M. Kovochich, M. Liong, H. Meng, S. Kabehiel, S. George, J.I. Zink, and A.E. Nel. 2009. Polyethleneimine coating enhances the cellular uptake of mesoporous silica nanoparticles and allows safe delivery of siRNA and DNA constructs. ACS Nano 3:3273-3286.

55.) Song, W.J., J.Z. Du, T.M. Sun, P.Z. Zhang, and J. Wang. 2010. Gold nanoparticles capped with polyethyleneimine for enhanced siRNA delivery. Small 6:239-246.

56.) Sherman, F. 1991. Guide to Yeast Genetics and Molecular Biology. Academic press Inc.

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