editorial board
past issues
contributors
feedback
contact us

 

printer friendly page    email page

2011, Vol. 6 No. 1, Article 74

 

Current Modes of Transfection – Emphasizing on nucleofection

M. D. Pratheesh*, N. Anandlaxmi1, C. Harish2,
R. Anoopraj3 and Justin Davis4

 

*Veterinary Physiology and Climatology Division
2,3Veterinary Pathology Division
4Veterinary Bacteriology Division
Indian Veterinary Research Institute, Izatnagar, Bareilly-UP

1Dairy Cattle Physiology Division
National Dairy Research Institute,
Karnal (Haryana), India-132001

 

*Corresponding Author; e-mail address: pratheeshmd@gmail.com

 


ABSTRACT

The process of introducing nucleic acids into eukaryotic cells by various methods is defined as Transfection. Using various chemical, lipid or physical methods, this gene transfer technology is a powerful tool for studying gene function in the context of a cell. Transfection is an essential tool for numerous in vitro applications including studies of gene expression, promoter analysis, and intracellular signaling pathways and also for therapeutic strategies such as tissue engineering and gene therapy.
Here we emphasize the use of nucleofection, a new, electroporation-based transfection method that enables the DNA to enter directly the nucleus, for the transfection of different cell types. The development of reporter gene systems and selection methods for stable maintenance and expression of transferred DNA have greatly expanded the applications for transfection.

KEY WORDS

Transfection, nucleofection, electroporation, lipofection, acoustic energy.

INTRODUCTION

Transfection is a molecular tool by which there can be deliberate modification of genetic make up of an animal by transferring in vitro recombined gene constructs to the genome (Brem et al. 1996). The main principle of gene transfer is to facilitate the transfer of heterologous DNA in to the nucleus of a target cells where its integration in to the host genome takes place. This technique can be utilized for the production of animals with better production performances. The universality of genetic code in animals theoretically allows the transfer of DNA between genetically unrelated species. When a gene construct is expressed it is called as transgene. The protein coded by this transgene is the transgenic product. Animals in which transgenes are integrated are called as transgenic animals and if the transgene is passed on to the offspring, transgenic lines or population will be created (Houdebine, 2003). Essentially, transfection is a method that neutralizes or obviates the issue of introducing negatively charged molecules (e.g., phosphate backbones of DNA and RNA) into cells with a negatively charged membrane.
Transfection is either transient or stable in nature. When foreign DNA is introduced, but is eliminated by the cell prior to or during mitosis, it is transient transfection. Much more rarely, foreign DNA is introduced and enters the cell nucleus, adding to or replacing a portion of the cell's native DNA. This type of transfection transforms the cell so that the DNA change is duplicated during mitosis, and is called stable transfection (Schulz et al. 2003).
The genetic engineer designs a transgene – containing the gene of interest plus promoter sequence and a corresponding poly A sequence. Many genes are only need to be expressed in particular tissue only, which is controlled by promoter sequence that is specially designed to ensure that the gene will function in the target tissue of the recipient animal. This is crucial, for example, when the gene of interest to be expressed in the milk of a mammal.

MODES OF GENE TRANSFER

To date broadly different modes of gene transfer systems are available for research workers:
(i) viral gene transfer (Pfeifer et al. 2002)
(ii) non-viral gene transfer (Gresch et al. 2004; Tinsley and Eriksson, 2004).
Viral method of transfection
Viral techniques are the most efficient systems to deliver DNA into cells (Georgievska et al. 2004). They generally achieve transgene expression in a high proportion of target cells. Viruses which have been modified for use in gene transfer include retrovirus (including lentivirus), herpes simplex virus, adenovirus and adeno-associated virus; AAV (Winkler, 2002). But viral vectors have many disadvantages mainly involving safety risks e.g. infections. In addition, the use of viral vectors such as adeno-associated virus, lentivirus or retrovirus requires each cDNA to be cloned into specific vectors, cDNAs must not be too long and these techniques are very demanding in methodological skills, time and laboratory safety precautions (Gresch et al. 2004).
At the same time, these methods suffer from several limitations such as time-consuming and laborious production of vectors, elevated laboratory costs due to high level of safety requirements, limitation of insert size and possible immunogenic reaction in clinical human trials. In summary, viral vectors currently applied in clinical research do not meet demands for a gene transfer safety (Gresch et al. 2004).
Non viral method of Transfection
There are two main categories of non-viral gene transfer techniques; physical and chemical.
The chemical methods include:
1. Liposome-based gene transfer or lipofection (Wu et al. 2000)
2. Calcium phosphate-mediated gene transfer (Chen and Okayama, 1988)
3. DEAE-dextran transfection technique (Pari and Xu, 2004)
4. Polyethyleneimine (PEI)-mediated gene delivery (Corso et al. 2005).
The physical methods include:
1. Electroporation (Gehl, 2003)
2. Ballistic gene transfer (introduces particles coated with DNA into cells) (Zhang et al. 2003; Wells, 2004)
3. Microinjection (DNA transfer through microcapillaries into cells) (Davis et al. 2000).
4. Nucleofection (Amaxa biosystem, 2004).
1. Chemical Reagents
One of the first chemical reagents used for transfer of nucleic acids into cultured mammalian cells was DEAE-dextran (McCutchan and Pagano,1968). DEAE-dextran is a cationic polymer that tightly associates with negatively charged nucleic acids. An excess of positive charge, contributed by the polymer in the DNA:polymer complex, allows the complex to come into closer association with the negatively charged cell membrane. Uptake of the complex is presumably by endocytosis. This method is successful for delivery of nucleic acids into cells for transient expression; that is, for short-term expression studies. Other synthetic cationic polymers have been used for the transfer of DNA into cells, including polybrene (Kawai and Nishizawa, 1984), polyethyleneimine (Boussif , 1995) and dendrimers (Kukowska-Latallo et al. 1996). Calcium phosphate co-precipitation became a popular transfection technique following the systematic examination of this method in the early 1970’s (Graham and van der Eb,1973). The protocol involves mixing DNA with calcium chloride, adding it in a controlled manner to a buffered saline/phosphate solution and allowing the mixture to incubate at room temperature. The controlled mixing generates a precipitate that is dispersed onto the cultured cells. The precipitate is taken up by the cells via endocytosis or phagocytosis. Calcium phosphate transfection is routinely used for both transient and stable transfection of a variety of cell types. Both chemical transfer methods are relatively inexpensive and can provide high efficiency of transfer in some cell types. However, these reagents can be quite toxic (particularly DEAE-dextran), and are prone to variability.
2. Electroporation
Electroporation is a physical process that transiently permeabilizes prokaryotic and eukaryotic cell membranes with an electrical pulse, thus permitting cell uptake of a wide variety of biological molecules (Gehl, 2003). Electroporation occurs as a result of the reorientation of lipid molecules of the bilayer membrane to form hydrophilic pores in the membrane. The distribution of such pores, both in terms of size and number, determine the electrical properties of the cell membrane. Changes in pore radius are effected by surface tension forces on the pore wall, diffusion of water molecules into and out of the pore and an electric field induced force of expansion. Major disadvantages of electroporation are the low transfection efficiency of primary cells and high cell mortality (Gresch et al. 2004).
3. Lipofection
Lipofection is one of the most widely used transfection method. Liposomes are small vesicles prepared from a suitable lipid. Initially, non-ionic lipids were used for preparing liposomes so that DNA had to be introduced within the vesicles following specific encapsulation and encapsidation procedures. The use of cationic lipids for the construction of liposomes has a distinct advantage as DNA spontaneously and efficiently complexes with these liposomes making encapsidation procedures unnecessary. The cationic liposomes have a single lipid bilayer membrane (unilamellar), and they bind to cells efficiently. They fuse with the plasma membrane and thereby deliver the DNA (complexed with them) into cells. Disadvantages of this method are quite low transfection efficiency in suspension cells and dependence on cell division as well as on high rate of endocytosis (Gresch et al. 2004).
4. Nucleofection
A highly effective non-viral method for gene transfer in to primary cells called nucleofection was developed by Amaxa Biosystems (Cologne, Germany (Gresch et al. 2004). This method is an electroporation-based method in which a combination of a specific nucleofector solution and patented specific electrical parameters achieve delivery of plasmid DNA into the cell nucleus. Cell-type specific combinations of electrical current and solutions make the technology unique in its ability to transfer polyanionic macromolecules directly into the nucleus. Therefore, even cells with limited potential to divide such as neurons are made accessible for efficient gene transfer (Gresch et al. 2004). Nucleofection is an efficient transfection technique for hMSCs which can serve as cellular vehicles for the delivery and local production of biological agents such as interleukin-12 (IL-12) (Aluigi et al. 2006). Efficient transfection of zebrafish fibroblasts by nucleofection with GFP gene was a non-viral technique of plasmid delivery (Badakov and Jaźwińska , 2006). Transfection of cultured Porcine embryonic fibroblast cells by nucleofection with Tg-EGFP offers a useful alternative for generation of genetically engineered pigs through nuclear transfer (Skrzyszowska et al. 2008). Nucleofection allows controlling protein amount expressed after transfection. Nucleofection is also more suitable for transfection with multiple genes, probably because of the direct transfer of a gene to the nucleus (Dityateva et al. 2003).
Advantages of nucleofection method include:
1. Easy, fast and safe to use.
2. Transfection efficiency is reproducible and allows biochemical assays to be performed.
3. Transfection is performed before cell plating and protein expression is usually observed 24 hours after transfection.
4. Long-term expression allows the study of protein function in later developmental processes such as protein/receptor trafficking, synaptogenes
Disadvantages include:
1. Large amounts of cells are needed for nucleofection.
2. Transfection efficiency depends on the cell type.
3. Success dependent on the type of gene construct used.
4. Decrease of expression can be observed in long-term experiments.
5. Early expression of some transgenes may interfere with cell attachment or with other developmental events preceding the process of interest.

ASSAY FOR TRANSFECTION

After the cells have been transfected, how will we determine success?. Plasmids containing reporter genes can be used easily to monitor transfection efficiencies and expression levels in the cells. An ideal reporter gene product is one that is unique to the cell, can be expressed from plasmid DNA and can be assayed conveniently. Generally, reporter gene assays are performed 1 - 3 days after transfection; the optimal time should be determined empirically.
Assay-based reporter technology, together with the availability of transfection reagents, provides the foundation for studying mammalian promoter and enhancer sequences, trans-acting proteins such as transcription factors, mRNA processing, protein:protein interactions, translation and recombination events .

REFERENCES

  1. Aluigi M, Fogli M, Curti A, Isidori A, Gruppioni E, Chiodoni C, Colombo M P, Versura P, D'Errico-Grigioni A, Ferri E, Baccarani M and Lemoli R M Nucleofection is an efficient nonviral transfection technique for human bone marrow-derived mesenchymal stem cells. J.Stem Cells.2006;24:454-461

  2. Badakov R and Jaźwińska A Efficient transfection of primary zebrafish fibroblasts by nucleofection. J. Cyto. Technol.2006 ; 51:105-110

  3. Brem G, Besenfelder U, Aigner B, Muller M , Liebl I, Schutz G, and Montoliu L YAC transgenesis in farm animals: rescue of albinism in rabbits. Mol. Reprod. Dev. 1996; 44:56-62

  4. Boussif O Proc. Natl. Acad. Sci. USA.1995; 92:7297-7304

  5. Cesnulevicius K, Marco T, Wesemann M, Thomas T, Barkhausen T, Grothe C Nucleofection Is the Most Efficient Nonviral Transfection Method for Neuronal Stem Cells Derived from Ventral Mesencephali with No Changes in Cell Composition or Dopaminergic Fate. Stem Cells 2006 24 : 2776 -2791

  6. Chen C A and Okayama H Calcium phosphate-mediated gene transfer: a highly efficient transfection system for stably transforming cells with plasmid DNA. Biotechniques1988: 6:632-638

  7. Corso T D, Torres G, Goulah C, Roy I, Gambino A S, Nayda J, Buckley T, Stachowiak E K, Bergey E J, Pudavar H, Dutta P, Bloom D C, Bowers W J and Stachowiak M K Assessment of viral and non-viral gene transfer into adult rat brains using HSV-1, calcium phosphate and PEI-based methods.J. Folia. Morphol. (Warsz) 2005 ; 64:130-144

  8. Davis B R, Brown D B, Prokopishyn N L and Yannariello-Brown J Micro-injection-mediated hematopoieticstem cell gene therapy. J. Curr. Opin. Mol .Therapy2000 ; 2: 412-419

  9. Distler Jörg H W, Astrid J, Mariola K, Michel B A , Renate G E , Steffen G and Oliver D Nucleofection: a new, highly efficient transfection method for primary human keratinocytes. J.Experimental Dermatology2005; 14: 315-320

  10. Dityateva G, Hammond M, Thiel C, Ruonala M O, Delling M, Siebenkotten G, Nix M and Dityatev A Rapid and efficient electroporation-based gene transfer into primary dissociated neurons. J. Neurosci. Methods2003; 130:65-73

  11. Gartner A, Collin L and Lalli G Nucleofection of primary neurons. Methods Enzymol. 2006; 406:374-388

  12. Gehl J Electroporation: theory and methods, perspectives for drug delivery, gene therapy and research.Acta. Physiol .Scand. 2003; 177: 437-447

  13. Georgievska B, Kirik D and Bjorklund A Overexpression of glial cell line-derived neurotrophic factor using a lentiviral vector induces time- and dose-dependent downregulation of tyrosine hydroxylase in the intact nigrostriatal dopamine system. J .Neurosci. 2004; 24: 6437-6445

  14. Graham F L and Van der Eb A J Virology1973 ; 52 :456-464

  15. Gresch O, Engel F B, Nesic D, Tran T T, England H M, Hickman E S, Korner I, Gan L, Chen S, Castro-Obregon S,Hammermann R, Wolf J, Muller-Hartmann H, Nix M, Siebenkotten G, Kraus G and Lun K New non-viral method for gene transfer into primary cells. Methods 2004; 33:151-163

  16. Guzman H R, Nguyen D X, Khan S and Prausnitz M R Ultrasound-mediated disruption of cell membranes. I.Quantification of molecular uptake and cell viability. J Acoust. Soc. Am. 2001;110: 588-596

  17. Houdebine L M Production of pharmaceutical proteins by transgenic animals. Comp Immunol. Microbiol. Infect. Dis. 2003; 32:107-121

  18. Kawai S and Nishizawa M Mol. Cell. Biol. 1984; 4;1172-1173

  19. Kobayashi N, Rivas-Carrillo J D, Soto-Gutierrez A, Fukazawa T, Chen Y, Navarro-Alvarez N and Tanaka N Gene delivery to embryonic stem cells. Birth Defects Res C Embryo Today 2005; 75:10-18

  20. Kukowska-Latallo J F Proc. Natl. Acad. Sci. USA 1996; 93: 4897-4902

  21. Lai W, Chang C H and Farber D L Gene transfection and expression in resting and activated murine CD4 Tcell subsets. J. Immunol .Methods 2004; 282:93-102

  22. Lakshmipathy U, Pelacho B, Sudo K, Linehan J L, Coucouvanis E, Kaufman D S and Verfaillie C M Efficient transfection of embryonic and adult stem cells. Stem Cells 2004; 22:531-543

  23. Leclere P G, Panjwani A, Docherty R, Berry M, Pizzey J and Tonge D A Effective gene delivery to adult neurons by a modified form of electroporation. J. Neurosci. Methods 2005; 142:137-143

  24. Liang H D, Lu Q L and Xue S A Optimisation of ultrasound-mediated gene transfer (sonoporation) in skeletalmuscle cells. Ultrasound Med. Biol2004; 30:1523-1529

  25. Liu J, Lewis T N and Prausnitz M R Non-invasive assessment and control of ultrasound-mediated membrane permeabilization. Pharm .Res 1998;15:918-924

  26. Lorenz P, Harnack U and Morgenstern R Efficient gene transfer into murine embryonic stem cells by nucleofection. Biotechnol .Lett 2004; 26:1589-1592

  27. Martinet W, Schrijvers D M and Kockx M M Nucleofection as an efficient nonviral transfection method for human monocytic cells. Biotechnol Lett 2004; 25:1025-1029

  28. Maasho K, Marusina A and Reynolds M N Efficient gene transfer into the human natural killer cell line, NKL, using the Amaxa nucleofection system. J. Immunol. Methods 2004; 284:133–140

  29. McCutchan J H and Pagano J S J. Natl.Cancer Inst. 1968; 41:351-352

  30. Miller D L and Gies R A Enhancement of ultrasonically-induced hemolysis by perfluorocarbon-based compared to airbasedecho-contrast agents. Ultrasound Med .Biol. 1998; 24:285-292

  31. Pari G S and Xu Y Gene transfer into mammalian cells using calcium phosphate and DEAE-dextran. Methods. Mol .Biol. 2004; 245:25-32

  32. Pfeifer A, Ikawa M, Dayn Y and Verma I M Transgenesis by lentiviral vectors: lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos . Proc. Natl. Acad. Sci .U S A2002; 99:2140-2145

  33. Schulz T C, Palmarini G M, Noggle S A, Weiler D A, Mitalipova M M and Condie B G Directed neuronal differentiation of human embryonic stem cells. BMC Neurosci 2003; 4:27-28

  34. Skrzyszowska M, Samiec M, Słomski R, Lipiński D and Mały E Development of porcine transgenic nuclear-transferred embryos derived from fibroblast cells transfected by the novel technique of nucleofection or standard lipofection Theriogenology 2008; 70: 248-259

  35. Tieleman D P, Leontiadou H, Mark A E and Marrink S J Simulation of pore formation in lipid bilayers by mechanicalstress and electric fields. J. Am. Chem. Soc 2003; 125:6382-6383

  36. Tinsley R and Eriksson P Use of gene therapy in central nervous system repair. Acta. Neurol. Scand . 2004;.109:1-8

  37. Wells D J Gene therapy progress and prospects: electroporation and other physical methods. Gene Therapy2004 ; 11:1363-1369

  38. Winkler C Toward a restorative therapy for Parkinson's disease. Studies on the functional efficacy of intracerebral fetal cell transplants and GDNF delivery. Ph D thesis.Lund University2002: 172

  39. Wu B Y, Liu R Y, So K L and Yu A C Multi-lipofection efficiently transfected genes into astrocytes in primary culture. J. Neurosci. Methods 2000;102 :133-141

  40. Zhang E P, Franke J, Schroff M, Junghans C, Wittig B and Hoffmann F Ballistic CTLA4 and IL-4 gene transfer into the lower lid prolongs orthotopic corneal graft survival in mice. Graefes Arch. Clin. Exp. Ophthalmol . 2003; 241:921-926


Table: Uptodate reports of Nucleofection studies in various cell types

  

SI No

Type of Cells

Gene/ plasmid

Reference

1

Human monocytic cell lines

Capped and poly adenylated mRNA for enhanced green fluorescent protein gene

Martinet  et al.  2004

2

Murine CD4 T cell

cDNA encoding green fluorescent protein (GFP)

Lai et al. 2004

3

Murine embryonic stem cells

Plasmid encoding enhanced green fluorescent protein (EGFP)

Lakshmipathy et al. 2004; Lorenz et al. 2004 )

4

Human embryonic stem cells

 

 

 

genes such as oct-4, sox-17, foxa2, mixl1, pdx-1, insulin 1, glucagons and somatostatin

 

Kobayashi et al. 2005

 

5

Retinal ganglionic cells (adult neuronal cells)

cDNA-encoding GFP

Leclere et al.  2005

 

6

Human bone marrow derived mesenchymal stem cells

GFP

 

 

Aluigi etal. 2006

7

human multipotent adipose tissue-derived stem (hMADS) cells

EGFP

Zaragosi et al. 2007

8

Mouse/ chicken hippocampal neural cells

GFP

Dityateva et al. 2003

9

Embryonic palatal mesenchymal (EPM) cells

plasmid pEGFP-N1

Gresch et al. 2004

10

Primary neurons

EGFP

Gartner et al. 2006

11

Natural killer cells (NK Cells)

EGFP

Maasho et al. 2004

12

Neuronal progenitor cells (NPCs)

EGFP

Cesnulevicius et al. 2006

13

Primary

epidermal keratinocytes

 

 

Distler et al. 2005



Copyright © Vet Scan 2005-

All Right Reserved with VetScan
www.vetscan.co.in www.kashvet.org
ISSN 0973-6980

 

Home | e-Learning |Resources | Alumni | Forum | Picture blog | Disclaimer

                   

 

 

powered by eMedia Services