Nucleofection is an

electroporation Electroporation, or electropermeabilization, is a microbiology technique in which an electrical field is applied to cells in order to increase the permeability of the cell membrane, allowing chemicals, drugs, electrode arrays or DNA to be introdu ...

-based

transfection Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. It may also refer to other methods and cell types, although other terms are often preferred: " transformation" is typically used to de ...

method which enables transfer of

nucleic acids Nucleic acids are biopolymers, macromolecules, essential to all known forms of life. They are composed of nucleotides, which are the monomers made of three components: a 5-carbon sugar, a phosphate group and a nitrogenous base. The two main cl ...

such as DNA and

RNA Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydra ...

into

cells Cell most often refers to: * Cell (biology), the functional basic unit of life Cell may also refer to: Locations * Monastic cell, a small room, hut, or cave in which a religious recluse lives, alternatively the small precursor of a monastery w ...

by applying a specific voltage and reagents. Nucleofection, also referred to as nucleofector technology, was invented by the biotechnology company Amaxa. "Nucleofector" and "nucleofection" are

trademarks A trademark (also written trade mark or trade-mark) is a type of intellectual property consisting of a recognizable sign, design, or expression that identifies products or services from a particular source and distinguishes them from ot ...

owned by Lonza Cologne AG, part of the

Lonza Group Lonza Group is a Swiss multinational manufacturing company for the pharmaceutical, biotechnology and nutrition sectors, headquartered in Basel, with major facilities in Europe, North America and South Asia. Lonza was established under that name ...

Applications

Nucleofection is a method to transfer substrates into mammalian cells so far considered difficult or even impossible to transfect. Examples for such substrates are nucleic acids, like the DNA of an isolated

gene In biology, the word gene (from , ; "... Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a b ...

cloned Cloning is the process of producing individual organisms with identical or virtually identical DNA, either by natural or artificial means. In nature, some organisms produce clones through asexual reproduction. In the field of biotechnology, ...

into a

plasmid A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; howev ...

, or

small interfering RNA Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA at first non-coding RNA molecules, typically 20-24 (normally 21) base pairs in length, similar to miRNA, and operating ...

(siRNA) for knocking down expression of a specific

endogenous Endogenous substances and processes are those that originate from within a living system such as an organism, tissue, or cell. In contrast, exogenous substances and processes are those that originate from outside of an organism. For example, ...

gene. Primary cells, for example

stem cell In multicellular organisms, stem cells are undifferentiated or partially differentiated cells that can differentiate into various types of cells and proliferate indefinitely to produce more of the same stem cell. They are the earliest type of ...

s, especially fall into this category, although many other

cell lines An immortalised cell line is a population of cells from a multicellular organism which would normally not proliferate indefinitely but, due to mutation, have evaded normal cellular senescence and instead can keep undergoing division. The cell ...

are also difficult to transfect. Primary cells are freshly isolated from

body tissue In biology, tissue is a biological organizational level between cells and a complete organ. A tissue is an ensemble of similar cells and their extracellular matrix from the same origin that together carry out a specific function. Organs are th ...

and thus cells are unchanged, closely resembling the

in-vivo Studies that are ''in vivo'' (Latin for "within the living"; often not italicized in English) are those in which the effects of various biological entities are tested on whole, living organisms or cells, usually animals, including humans, and pl ...

situation, and are therefore of particular relevance for

medical research Medical research (or biomedical research), also known as experimental medicine, encompasses a wide array of research, extending from " basic research" (also called ''bench science'' or ''bench research''), – involving fundamental scienti ...

purposes. In contrast, cell lines have often been cultured for decades and may significantly differ from their origin.

Mechanism

Based on the physical method of

, nucleofection uses a combination of electrical parameters, generated by a device called Nucleofector, with cell-type specific reagents. The substrate is transferred directly into the cell nucleus and the

cytoplasm In cell biology, the cytoplasm is all of the material within a eukaryotic cell, enclosed by the cell membrane, except for the cell nucleus. The material inside the nucleus and contained within the nuclear membrane is termed the nucleoplasm. ...

. In contrast, other commonly used non-viral transfection methods rely on

cell division Cell division is the process by which a parent cell divides into two daughter cells. Cell division usually occurs as part of a larger cell cycle in which the cell grows and replicates its chromosome(s) before dividing. In eukaryotes, there ar ...

for the transfer of DNA into the nucleus. Thus, nucleofection provides the ability to transfect even non-dividing cells, such as

neuron A neuron, neurone, or nerve cell is an membrane potential#Cell excitability, electrically excitable cell (biology), cell that communicates with other cells via specialized connections called synapses. The neuron is the main component of nervous ...

and resting

blood cells A blood cell, also called a hematopoietic cell, hemocyte, or hematocyte, is a cell produced through hematopoiesis and found mainly in the blood. Major types of blood cells include red blood cells (erythrocytes), white blood cells (leukocytes), ...

. Before the introduction of the Nucleofector Technology, efficient

gene transfer Horizontal gene transfer (HGT) or lateral gene transfer (LGT) is the movement of genetic material between Unicellular organism, unicellular and/or multicellular organisms other than by the ("vertical") transmission of DNA from parent to offsprin ...

into primary cells had been restricted to the use of

viral vector Viral vectors are tools commonly used by molecular biologists to deliver genetic material into cells. This process can be performed inside a living organism ('' in vivo'') or in cell culture (''in vitro''). Viruses have evolved specialized mole ...

s, which typically involve disadvantages such as safety risks, lack of reliability, and high cost. The non-viral gene transfer methods available were not suitable for the efficient transfection of primary cells. Non-viral delivery methods may require cell division for completion of transfection, since the DNA enters the nucleus during breakdown of the

nuclear envelope The nuclear envelope, also known as the nuclear membrane, is made up of two lipid bilayer membranes that in eukaryotic cells surround the nucleus, which encloses the genetic material. The nuclear envelope consists of two lipid bilayer membrane ...

upon

or by a specific localization sequence. Optimal nucleofection conditions depend upon the individual

cell type A cell type is a classification used to identify cells that share morphological or phenotypical features. A multicellular organism may contain cells of a number of widely differing and specialized cell types, such as muscle cells and skin cells ...

, not on the substrate being transfected. This means that identical conditions are used for the nucleofection of DNA, RNA, siRNAs,

shRNA A short hairpin RNA or small hairpin RNA (shRNA/Hairpin Vector) is an artificial RNA molecule with a tight hairpin turn that can be used to silence target gene expression via RNA interference (RNAi). Expression of shRNA in cells is typically acco ...

mRNA In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of synthesizing a protein. mRNA is created during the ...

s and pre-mRNAs, BACs,

peptide Peptides (, ) are short chains of amino acids linked by peptide bonds. Long chains of amino acids are called proteins. Chains of fewer than twenty amino acids are called oligopeptides, and include dipeptides, tripeptides, and tetrapeptides. ...

morpholino A Morpholino, also known as a Morpholino oligomer and as a phosphorodiamidate Morpholino oligomer (PMO), is a type of oligomer molecule (colloquially, an oligo) used in molecular biology to modify gene expression. Its molecular structure contains ...

s, PNA, or other biologically active molecules.

References

* * * {{cite journal, title=Nucleofection is an efficient non-viral transfection technique for human bone marrow-derived mesenchymal stem cells, journal=Stem Cells, volume=24, number=2, date=February 2006, pages=454–461, authors=Michela Aluigi, Miriam Fogli, Antonio Curti, Alessandro Isidori, Elisa Gruppioni, Claudia Chiodoni, Mario P. Colombo, Piera Versura, Antonia D’Errico-Grigioni, Elisa Ferri, Michele Baccarani, and Roberto M. Lemoli, pmid=16099993, doi=10.1634/stemcells.2005-0198, doi-access=free Molecular biology Genetic engineering

Applications

Mechanism

See also

References