Nucleofection
Encyclopedia
Nucleofection refers to electroporation, a transfection
method which enables transfer of nucleic acids such as DNA
, RNA
, Small interfering RNA
into cells
. Nucleofection, also referred to as Nucleofector Technology, was invented by the biotechnology company amaxa. “Nucleofector” and “nucleofection” are trademarks, owned by Lonza Cologne AG - part of the Lonza Group
.
so far considered difficult or even impossible to transfect. Examples for such substrates are nucleic acids, like the DNA of an isolated gene
cloned
into a plasmid
or Small interfering RNA for knocking down expression of a specific endogenous
gene.
Primary cells, for example stem cell
s, especially fall into this category, although many other cell lines
are also difficult to transfect. Primary cells are freshly isolated from body tissue
and thus cells are unchanged, closely resembling the in-vivo situation, and are therefore of particular relevance for medical research
purposes. In contrast, cell lines have often been cultured for decades and may significantly differ from their origin.
, 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 contrast, other commonly used non-viral transfection methods rely on cell division
for the transfer of DNA into the nucleus. Thus, nucleofection provides the ability to transfect even non-dividing cells, such as neuron and resting blood cells. Before the introduction of the Nucleofector Technology, efficient gene transfer into primary cells had been restricted to the use of viral vector
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. All of the non-viral methods around required cell division for completion of transfection, since the DNA only enters the nucleus to a sufficient level during breakdown of the nuclear envelope
upon cell division
.
Optimal nucleofection conditions depend upon the individual cell type, not on the substrate being transfected. This means that identical conditions are used for the nucleofection of DNA, RNA, siRNAs, shRNAs, mRNAs and pre-mRNAs
, BACs
, peptide
s, morpholino
s, PNA, or other biologically active molecules.
Transfection
Transfection is the process of deliberately introducing nucleic acids into cells. The term is used notably for non-viral methods in eukaryotic cells...
method which enables transfer of nucleic acids such as DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
, RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
, Small interfering RNA
Sírna
Sírna Sáeglach , son of Dian mac Demal, son of Demal mac Rothechtaid, son of Rothechtaid mac Main, was, according to medieval Irish legend and historical tradition, a High King of Ireland...
into cells
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
. Nucleofection, also referred to as Nucleofector Technology, was invented by the biotechnology company amaxa. “Nucleofector” and “nucleofection” are trademarks, owned by Lonza Cologne AG - part of the Lonza Group
Lonza Group
Lonza Group is a Swiss chemicals and biotechnology company, headquartered in Basel. The firm provides a number of products and services to the pharmaceutical and life science industries, including organic fine and performance chemicals, custom manufacturing of biopharmaceuticals, chemical synthesis...
.
Applications
Nucleofection is a method to transfer substrates into mammalian cellsMammal
Mammals are members of a class of air-breathing vertebrate animals characterised by the possession of endothermy, hair, three middle ear bones, and mammary glands functional in mothers with young...
so far considered difficult or even impossible to transfect. Examples for such substrates are nucleic acids, like the DNA of an isolated gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
cloned
Molecular cloning
Molecular cloning refers to a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms...
into a plasmid
Plasmid
In microbiology and genetics, a plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double-stranded and, in many cases, circular...
or Small interfering RNA for knocking down expression of a specific endogenous
Endogenous
Endogenous substances are those that originate from within an organism, tissue, or cell. Endogenous retroviruses are caused by ancient infections of germ cells in humans, mammals and other vertebrates...
gene.
Primary cells, for example stem cell
Stem cell
This article is about the cell type. For the medical therapy, see Stem Cell TreatmentsStem cells are biological cells found in all multicellular organisms, that can divide and differentiate into diverse specialized cell types and can self-renew to produce more stem cells...
s, especially fall into this category, although many other cell lines
Cell culture
Cell culture is the complex process by which cells are grown under controlled conditions. In practice, the term "cell culture" has come to refer to the culturing of cells derived from singlecellular eukaryotes, especially animal cells. However, there are also cultures of plants, fungi and microbes,...
are also difficult to transfect. Primary cells are freshly isolated from body tissue
Tissue (biology)
Tissue is a cellular organizational level intermediate between cells and a complete organism. A tissue is an ensemble of cells, not necessarily identical, but from the same origin, that together carry out a specific function. These are called tissues because of their identical functioning...
and thus cells are unchanged, closely resembling the in-vivo situation, and are therefore of particular relevance for medical research
Biomedical research
Biomedical research , in general simply known as medical research, is the basic research, applied research, or translational research conducted to aid and support the body of knowledge in the field of medicine...
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 electroporationElectroporation
Electroporation, or electropermeabilization, is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field...
, 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
Cell nucleus
In cell biology, the nucleus is a membrane-enclosed organelle found in eukaryotic cells. It contains most of the cell's genetic material, organized as multiple long linear DNA molecules in complex with a large variety of proteins, such as histones, to form chromosomes. The genes within these...
and the cytoplasm
Cytoplasm
The cytoplasm is a small gel-like substance residing between the cell membrane holding all the cell's internal sub-structures , except for the nucleus. All the contents of the cells of prokaryote organisms are contained within the cytoplasm...
. In contrast, other commonly used non-viral transfection methods rely on cell division
Cell division
Cell division is the process by which a parent cell divides into two or more daughter cells . Cell division is usually a small segment of a larger cell cycle. This type of cell division in eukaryotes is known as mitosis, and leaves the daughter cell capable of dividing again. The corresponding sort...
for the transfer of DNA into the nucleus. Thus, nucleofection provides the ability to transfect even non-dividing cells, such as neuron and resting blood cells. Before the introduction of the Nucleofector Technology, efficient gene transfer into primary cells had been restricted to the use of viral vector
Viral vector
Viral vectors are a tool commonly used by molecular biologists to deliver genetic material into cells. This process can be performed inside a living organism or in cell culture . Viruses have evolved specialized molecular mechanisms to efficiently transport their genomes inside the cells they infect...
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. All of the non-viral methods around required cell division for completion of transfection, since the DNA only enters the nucleus to a sufficient level during breakdown of the nuclear envelope
Nuclear envelope
A nuclear envelope is a double lipid bilayer that encloses the genetic material in eukaryotic cells. The nuclear envelope also serves as the physical barrier, separating the contents of the nucleus from the cytosol...
upon cell division
Cell division
Cell division is the process by which a parent cell divides into two or more daughter cells . Cell division is usually a small segment of a larger cell cycle. This type of cell division in eukaryotes is known as mitosis, and leaves the daughter cell capable of dividing again. The corresponding sort...
.
Optimal nucleofection conditions depend upon the individual cell type, not on the substrate being transfected. This means that identical conditions are used for the nucleofection of DNA, RNA, siRNAs, shRNAs, mRNAs and pre-mRNAs
Precursor mRNA
Precursor mRNA is an immature single strand of messenger ribonucleic acid . pre-mRNA is synthesized from a DNA template in the cell nucleus by transcription. Pre-mRNA comprises the bulk of heterogeneous nuclear RNA...
, BACs
Bacterial artificial chromosome
A bacterial artificial chromosome is a DNA construct, based on a functional fertility plasmid , used for transforming and cloning in bacteria, usually E. coli. F-plasmids play a crucial role because they contain partition genes that promote the even distribution of plasmids after bacterial cell...
, peptide
Peptide
Peptides are short polymers of amino acid monomers linked by peptide bonds. They are distinguished from proteins on the basis of size, typically containing less than 50 monomer units. The shortest peptides are dipeptides, consisting of two amino acids joined by a single peptide bond...
s, morpholino
Morpholino
In molecular biology, a Morpholino is a molecule in a particular structural family that is used to modify gene expression. Morpholino oligomers are an antisense technology used to block access of other molecules to specific sequences within nucleic acid...
s, PNA, or other biologically active molecules.