Editing Genetic transformation (section)

==Methods and mechanisms of transformation in laboratory ==
[[File:Artificial Bacterial Transformation.svg|thumb|391x391px|Schematic of bacterial transformation – for which artificial competence must first be induced]]

===Bacterial===
Artificial competence can be induced in laboratory procedures that involve making the cell passively permeable to DNA by exposing it to conditions that do not normally occur in nature.<ref>{{cite periodical |vauthors=Donahue RA, Bloom FR |title=Large-volume transformation with high-throughput efficiency chemically competent cells |periodical=Focus |volume=20 |issue=2 |pages=54–56 |date=July 1998 |url=http://www.invitrogen.com/etc/medialib/en/filelibrary/pdf/focus.Par.78703.File.dat/Focus%20Volume%2020%20Issue%202.pdf |archive-url=https://web.archive.org/web/20130306120401/http://www.invitrogen.com/etc/medialib/en/filelibrary/pdf/focus.Par.78703.File.dat/Focus%20Volume%2020%20Issue%202.pdf |archive-date=2013-03-06 |oclc=12352630 |via=Invitrogen}}{{rs|date=September 2022|reason=corporate publication where the authors work for the corporation;}}</ref> Typically the cells are incubated in a solution containing [[divalent]] [[cation]]s (often [[calcium chloride]]) under cold conditions, before being exposed to a heat pulse (heat shock). Calcium chloride partially disrupts the cell membrane, which allows the recombinant DNA to enter the host cell. Cells that are able to take up the DNA are called competent cells.

It has been found that growth of Gram-negative bacteria in 20 mM Mg reduces the number of protein-to-[[lipopolysaccharide]] bonds by increasing the ratio of ionic to covalent bonds, which increases membrane fluidity, facilitating transformation.<ref name="Srivastava">{{cite book | last = Srivastava | first = Sheela | name-list-style = vanc | title = Genetics of Bacteria | publisher = [[Springer-Verlag]] | year = 2013 | location = India | url = https://link.springer.com/content/pdf/10.1007%2F978-81-322-1090-0.pdf#page=112| doi = 10.1007/978-81-322-1090-0 | isbn = 978-81-322-1089-4 | s2cid = 35917467 }}</ref> The role of lipopolysaccharides here are verified from the observation that shorter O-side chains are more effectively transformed – perhaps because of improved DNA accessibility.

The surface of bacteria such as ''E. coli'' is negatively charged due to [[phospholipids]] and [[lipopolysaccharide]]s on its cell surface, and the DNA is also negatively charged. One function of the divalent cation therefore would be to shield the charges by coordinating the phosphate groups and other negative charges, thereby allowing a DNA molecule to adhere to the cell surface.

DNA entry into ''E. coli'' cells is through channels known as zones of adhesion or Bayer's junction, with a typical cell carrying as many as 400 such zones. Their role was established when [[cobalamine]] (which also uses these channels) was found to competitively inhibit DNA uptake. Another type of channel implicated in DNA uptake consists of poly (HB):poly P:Ca. In this poly (HB) is envisioned to wrap around DNA (itself a polyphosphate), and is carried in a shield formed by Ca ions.<ref name="Srivastava" />

It is suggested that exposing the cells to divalent cations in cold condition may also change or weaken the cell surface structure, making it more permeable to DNA.  The heat-pulse is thought to create a thermal imbalance across the cell membrane, which forces the DNA to enter the cells through either cell pores or the damaged cell wall.

[[Electroporation]] is another method of promoting competence.  In this method the cells are briefly shocked with an [[electric field]] of 10-20 [[Volt|kV]]/cm, which is thought to create holes in the cell membrane through which the plasmid DNA may enter.  After the electric shock, the holes are rapidly closed by the cell's membrane-repair mechanisms.

===Yeast===
Most species of [[yeast]], including ''[[Saccharomyces cerevisiae]]'', may be transformed by exogenous DNA in the environment. Several methods have been developed to facilitate this transformation at high frequency in the lab.<ref>{{cite journal | vauthors = Kawai S, Hashimoto W, Murata K | title = Transformation of Saccharomyces cerevisiae and other fungi: methods and possible underlying mechanism | journal = Bioengineered Bugs | volume = 1 | issue = 6 | pages = 395–403 | date = 1 November 2010 | pmid = 21468206 | doi = 10.4161/bbug.1.6.13257 | pmc=3056089}}</ref>
* Yeast cells may be treated with enzymes to degrade their cell walls, yielding [[spheroplast]]s. These cells are very fragile but take up foreign DNA at a high rate.<ref>{{cite journal | vauthors = Hinnen A, Hicks JB, Fink GR | title = Transformation of yeast | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 75 | issue = 4 | pages = 1929–33 | date = April 1978 | pmid = 347451 | pmc = 392455 | doi = 10.1073/pnas.75.4.1929 | bibcode = 1978PNAS...75.1929H | doi-access = free }}</ref>
* Exposing intact yeast cells to [[alkali]] [[cation]]s such as those of [[caesium]] or [[lithium]] allows the cells to take up plasmid DNA.<ref>{{cite journal | vauthors = Ito H, Fukuda Y, Murata K, Kimura A | title = Transformation of intact yeast cells treated with alkali cations | journal = Journal of Bacteriology | volume = 153 | issue = 1 | pages = 163–8 | date = January 1983 | doi = 10.1128/JB.153.1.163-168.1983 | pmid = 6336730 | pmc = 217353 }}</ref> Later protocols adapted this transformation method, using [[lithium acetate]], [[polyethylene glycol]], and single-stranded DNA.<ref>{{cite book | vauthors = Gietz RD, Woods RA | chapter = Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method | volume = 350 | pages = 87–96 | year = 2002 | pmid = 12073338 | doi = 10.1016/S0076-6879(02)50957-5 | isbn = 9780121822538 | series = Methods in Enzymology | title = Guide to Yeast Genetics and Molecular and Cell Biology - Part B }}</ref> In these protocols, the single-stranded DNA preferentially binds to the yeast cell wall, preventing plasmid DNA from doing so and leaving it available for transformation.<ref>{{cite journal | vauthors = Gietz RD, Schiestl RH, Willems AR, Woods RA | title = Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure | journal = Yeast | volume = 11 | issue = 4 | pages = 355–60 | date = April 1995 | pmid = 7785336 | doi = 10.1002/yea.320110408 | s2cid = 22611810 }}</ref>
* [[Electroporation]]: Formation of transient holes in the cell membranes using electric shock; this allows DNA to enter as described above for bacteria.<ref name=":0">{{cite journal|last=Schiestl|first=Robert H.|author2=Manivasakam, P. |author3=Woods, Robin A. |author4= Gietzt, R.Daniel |title=Introducing DNA into Yeast by Transformation|journal=Methods|date=1 August 1993|volume=5|issue=2|pages=79–85|doi=10.1006/meth.1993.1011}}</ref>
* Enzymatic digestion<ref>{{cite journal|last=Spencer|first=F.|author2=Ketner, G. |author3=Connelly, C. |author4= Hieter, P. |title=Targeted Recombination-Based Cloning and Manipulation of Large DNA Segments in Yeast|journal=Methods|date=1 August 1993|volume=5|issue=2|pages=161–175|doi=10.1006/meth.1993.1021}}</ref> or agitation with glass beads<ref>{{cite journal | vauthors = Costanzo MC, Fox TD | title = Transformation of yeast by agitation with glass beads | journal = Genetics | volume = 120 | issue = 3 | pages = 667–70 | date = November 1988 | doi = 10.1093/genetics/120.3.667 | pmid = 3066683 | pmc = 1203545 }}</ref> may also be used to transform yeast cells.

'''Efficiency''' &ndash; Different yeast genera and species take up foreign DNA with different efficiencies.<ref name="Dohmen1991">{{cite journal | vauthors = Dohmen RJ, Strasser AW, Höner CB, Hollenberg CP | title = An efficient transformation procedure enabling long-term storage of competent cells of various yeast genera | journal = Yeast | volume = 7 | issue = 7 | pages = 691–2 | date = October 1991 | pmid = 1776359 | doi = 10.1002/yea.320070704 | s2cid = 7108750 }}</ref> Also, most transformation protocols have been developed for baker's yeast, ''S. cerevisiae'', and thus may not be optimal for other species. Even within one species, different strains have different transformation efficiencies, sometimes different by three orders of magnitude. For instance, when S. cerevisiae strains were transformed with 10&nbsp;ug of plasmid YEp13, the strain DKD-5D-H yielded between 550 and 3115 colonies while strain OS1 yielded fewer than five colonies.<ref name="Hayama2002">{{cite journal | vauthors = Hayama Y, Fukuda Y, Kawai S, Hashimoto W, Murata K | title = Extremely simple, rapid and highly efficient transformation method for the yeast Saccharomyces cerevisiae using glutathione and early log phase cells | journal = Journal of Bioscience and Bioengineering | volume = 94 | issue = 2 | pages = 166–71 | year = 2002 | pmid = 16233287 | doi=10.1016/s1389-1723(02)80138-4}}</ref>

===Plants===
<!-- This section is linked from [[Arabidopsis thaliana]] -->
A number of methods are available to transfer DNA into plant cells. Some [[vector (molecular biology)|vector]]-mediated methods are:
*''[[Agrobacterium]]''-mediated transformation is the easiest and most simple plant transformation. Plant tissue (often leaves) are cut into small pieces, e.g. 10x10mm, and soaked for ten minutes in a fluid containing suspended ''Agrobacterium''. The bacteria will attach to many of the plant cells exposed by the cut. The plant cells secrete wound-related phenolic compounds which in turn act to upregulate the virulence operon of the Agrobacterium. The virulence operon includes many genes that encode for proteins that are part of a Type IV secretion system that exports from the bacterium proteins and DNA (delineated by specific recognition motifs called border sequences and excised as a single strand from the virulence plasmid) into the plant cell through a structure called a pilus. The transferred DNA (called T-DNA) is piloted to the plant cell nucleus by nuclear localization signals present in the Agrobacterium protein VirD2, which is covalently attached to the end of the T-DNA at the Right border (RB). Exactly how the T-DNA is integrated into the host plant genomic DNA is an active area of plant biology research. Assuming that a selection marker (such as an antibiotic resistance gene) was included in the T-DNA, the transformed plant tissue can be cultured on selective media to produce shoots. The shoots are then transferred to a different medium to promote root formation. Once roots begin to grow from the transgenic shoot, the plants can be transferred to soil to complete a normal life cycle (make seeds). The seeds from this first plant (called the T1, for first transgenic generation) can be planted on a selective (containing an antibiotic), or if an [[herbicide resistance]] gene was used, could alternatively be planted in soil, then later treated with herbicide to kill wildtype segregants. Some plants species, such as ''Arabidopsis thaliana'' can be transformed by dipping the flowers or whole plant, into a suspension of ''Agrobacterium tumefaciens'', typically strain C58 (C=Cherry, 58=1958, the year in which this particular strain of ''A. tumefaciens'' was isolated from a cherry tree in an orchard at Cornell University in Ithaca, New York). Though many plants remain recalcitrant to transformation by this method, research is ongoing that continues to add to the list the species that have been successfully modified in this manner.
*[[Viral transformation]] ([[transduction (genetics)|transduction]]): Package the desired genetic material into a suitable plant virus and allow this modified virus to infect the plant. If the genetic material is DNA, it can recombine with the chromosomes to produce transformant cells. However, genomes of most plant viruses consist of single stranded [[RNA]] which replicates in the cytoplasm of infected cell. For such genomes this method is a form of [[transfection]] and not a real transformation, since the inserted genes never reach the nucleus of the cell and do not integrate into the host genome. The progeny of the infected plants is virus-free and also free of the inserted gene.

Some vector-less methods include:
*[[Gene gun]]: Also referred to as particle bombardment, microprojectile bombardment, or biolistics. Particles of gold or tungsten are coated with DNA and then shot into young plant cells or plant embryos. Some genetic material will stay in the cells and transform them. This method also allows transformation of plant plastids. The [[transformation efficiency]] is lower than in ''Agrobacterium''-mediated transformation, but most plants can be transformed with this method.
*[[Electroporation]]: Formation of transient holes in cell membranes using electric pulses of high field strength; this allows DNA to enter as described above for bacteria.<ref name="ISC BIOLOGY">{{cite book | title=ISC BIOLOGY | publisher=Nageen Prakashan | author=V.Singh and D.K.Jain | chapter=Applications of recombinant DNA | year=2014 | pages=840}}</ref>

=== Fungi ===
There are some methods to produce transgenic [[Fungus|fungi]] most of them being analogous to those used for plants. However, fungi have to be treated differently due to some of their microscopic and biochemical traits:

* A major issue is the [[Dikaryon|dikaryotic state]] that parts of some fungi are in; dikaryotic cells contain two haploid nuclei, one of each parent fungus. If only one of these gets transformed, which is the rule, the percentage of transformed nuclei decreases after each [[Spore|sporulation]].<ref name=":1">{{Cite journal|last1=Poyedinok|first1=N. L.|last2=Blume|first2=Ya. B.|date=March 2018|title=Advances, Problems, and Prospects of Genetic Transformation of Fungi|journal=Cytology and Genetics|volume=52|issue=2|pages=139–154|doi=10.3103/S009545271802007X|s2cid=4561837|issn=0095-4527}}</ref>
* Fungal cell walls are quite thick hindering DNA uptake so (partial) removal is often required;<ref>{{Cite journal|last1=He|first1=Liya|last2=Feng|first2=Jiao|last3=Lu|first3=Sha|last4=Chen|first4=Zhiwen|last5=Chen|first5=Chunmei|last6=He|first6=Ya|last7=Yi|first7=Xiuwen|last8=Xi|first8=Liyan|date=2017|title=Genetic transformation of fungi|journal=The International Journal of Developmental Biology|volume=61|issue=6–7|pages=375–381|doi=10.1387/ijdb.160026lh|pmid=27528043|issn=0214-6282|doi-access=free}}</ref> complete degradation, which is sometimes necessary,<ref name=":1" /> yields [[protoplast]]s.
* Mycelial fungi consist of filamentous [[hypha]]e, which are, if at all, separated by internal cell walls interrupted by pores big enough to enable nutrients and organelles, sometimes even nuclei, to travel through each hypha. As a result, individual cells usually cannot be separated. This is problematic as neighbouring transformed cells may render untransformed ones immune to selection treatments, e.g. by delivering nutrients or proteins for antibiotic resistance.<ref name=":1" />
* Additionally, growth (and thereby mitosis) of these fungi exclusively occurs at the tip of their hyphae which can also deliver issues.<ref name=":1" />

As stated earlier, an array of methods used for plant transformation do also work in fungi:

* Agrobacterium is not only capable of infecting plants but also fungi, however, unlike plants, fungi do not secrete the phenolic compounds necessary to trigger Agrobacterium so that they have to be added, e.g. in the form of [[acetosyringone]].<ref name=":1" />
* Thanks to development of an expression system for small RNAs in fungi the introduction of a [[CRISPR/Cas9-mediated genome editing|CRISPR/CAS9-system]] in fungal cells became possible.<ref name=":1" /> In 2016 the USDA declared that it will not regulate a white button mushroom strain edited with CRISPR/CAS9 to prevent fruit body browning causing a broad discussion about placing CRISPR/CAS9-edited crops on the market.<ref>{{Cite journal|last=Waltz|first=Emily|date=April 2016|title=Gene-edited CRISPR mushroom escapes US regulation|journal=Nature|volume=532|issue=7599|pages=293|doi=10.1038/nature.2016.19754|pmid=27111611|issn=0028-0836|bibcode=2016Natur.532..293W|doi-access=free}}</ref>
* Physical methods like electroporation, biolistics ("gene gun"), [[sonoporation]] that uses cavitation of gas bubbles produced by ultrasound to penetrate the cell membrane, etc. are also applicable to fungi.<ref>{{Cite journal|last1=Rivera|first1=Ana Leonor|last2=Magaña-Ortíz|first2=Denis|last3=Gómez-Lim|first3=Miguel|last4=Fernández|first4=Francisco|last5=Loske|first5=Achim M.|date=June 2014|title=Physical methods for genetic transformation of fungi and yeast|journal=Physics of Life Reviews|volume=11|issue=2|pages=184–203|doi=10.1016/j.plrev.2014.01.007|pmid=24507729|bibcode=2014PhLRv..11..184R}}</ref>

===Animals===
Introduction of DNA into animal cells is usually called [[transfection]], and is discussed in the corresponding article.