Nucleofection is an

electroporation Electroporation, also known as electropermeabilization, is a microbiological and biotechnological technique in which an electric field is applied to cells to briefly increase the permeability of the cell membrane. The application of a high-vo ...

-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 des ...

method which enables transfer of

nucleic acids Nucleic acids are large biomolecules that are crucial in all cells and viruses. They are composed of nucleotides, which are the monomer components: a 5-carbon sugar, a phosphate group and a nitrogenous base. The two main classes of nucleic a ...

such as

DNA Deoxyribonucleic acid (; DNA) is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of al ...

and

RNA Ribonucleic acid (RNA) is a polymeric molecule that is essential for most biological functions, either by performing the function itself (non-coding RNA) or by forming a template for the production of proteins (messenger RNA). RNA and deoxyrib ...

into

cells Cell most often refers to: * Cell (biology), the functional basic unit of life * Cellphone, a phone connected to a cellular network * Clandestine cell, a penetration-resistant form of a secret or outlawed organization * Electrochemical cell, a d ...

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 form of intellectual property that consists of a word, phrase, symbol, design, or a combination that identifies a product or service from a particular source and distinguishes it from ot ...

owned by Lonza Cologne AG, part of the

Lonza Group Lonza Group AG 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 has two meanings. The Mendelian gene is a basic unit of heredity. The molecular gene is a sequence of nucleotides in DNA that is transcribed to produce a functional RNA. There are two types of molecular genes: protei ...

cloned Cloning is the process of producing individual organisms with identical genomes, either by natural or artificial means. In nature, some organisms produce clones through asexual reproduction; this reproduction of an organism by itself without ...

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 and ...

, or

small interfering RNA Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA, double-stranded non-coding RNA, non-coding RNA, RNA molecules, typically 20–24 base pairs in length, similar to microR ...

(siRNA) for knocking down expression of a specific

endogenous Endogeny, in biology, refers to the property of originating or developing from within an organism, tissue, or cell. For example, ''endogenous substances'', and ''endogenous processes'' are those that originate within a living system (e.g. an ...

gene. Primary cells, for example

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

s, especially fall into this category, although many other

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

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

body tissue In biology, tissue is an assembly of similar cells and their extracellular matrix from the same embryonic origin that together carry out a specific function. Tissues occupy a biological organizational level between cells and a complete org ...

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 ...

situation, and are therefore of particular relevance for

medical research Medical research (or biomedical research), also known as health research, refers to the process of using scientific methods with the aim to produce knowledge about human diseases, the prevention and treatment of illness, and the promotion of ...

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 The cell nucleus (; : nuclei) is a membrane-bound organelle found in eukaryote, eukaryotic cell (biology), cells. Eukaryotic cells usually have a single nucleus, but a few cell types, such as mammalian red blood cells, have #Anucleated_cells, ...

and the

cytoplasm The cytoplasm describes all the material within a eukaryotic or prokaryotic cell, enclosed by the cell membrane, including the organelles and excluding the nucleus in eukaryotic cells. The material inside the nucleus of a eukaryotic cell a ...

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

cell division Cell division is the process by which a parent cell (biology), 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 eukar ...

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

neuron A neuron (American English), neurone (British English), or nerve cell, is an membrane potential#Cell excitability, excitable cell (biology), cell that fires electric signals called action potentials across a neural network (biology), neural net ...

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 organisms other than by the ("vertical") transmission of DNA from parent to offspring (reproduction). HGT is an important factor in the e ...

into primary cells had been restricted to the use of

viral vector A viral vector is a modified virus designed to gene delivery, deliver genetic material into cell (biology), cells. This process can be performed inside an organism or in cell culture. Viral vectors have widespread applications in basic research, ...

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 membran ...

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 cell ...

, 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 acc ...

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 Protein biosynthesis, synthesizing a protein. mRNA is ...

s and pre-mRNAs, BACs,

peptide Peptides are short chains of amino acids linked by peptide bonds. A polypeptide is a longer, continuous, unbranched peptide chain. Polypeptides that have a molecular mass of 10,000 Da or more are called proteins. Chains of fewer than twenty am ...

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, author=Michela Aluigi , author2=Miriam Fogli , author3=Antonio Curti , author4=Alessandro Isidori , author5=Elisa Gruppioni , author6=Claudia Chiodoni , author7=Mario P. Colombo , author8=Piera Versura , author9=Antonia D'Errico-Grigioni , author10=Elisa Ferri , author11=Michele Baccarani , author12=Roberto M. Lemoli , pmid=16099993, doi=10.1634/stemcells.2005-0198, doi-access=free Molecular biology Genetic engineering

Applications

Mechanism

See also

References