Editing Genetic engineering (section)

=== Research ===

[[File:PCWmice1.jpg|thumb|[[Knockout mouse|Knockout mice]]]]

[[File:Expression of Human Wild-Type and P239S Mutant Palladin.png|thumb|Human cells in which some proteins are fused with [[green fluorescent protein]] to allow them to be visualised]]

Genetic engineering is an important tool for [[natural scientists]], with the creation of transgenic organisms one of the most important tools for analysis of gene function.<ref>{{cite book | vauthors = Praitis V, Maduro MF | title = Caenorhabditis elegans: Molecular Genetics and Development | chapter = Transgenesis in C. elegans | series = Methods in Cell Biology | volume = 106 | pages = 161–85 | date = 2011 | pmid = 22118277 | doi = 10.1016/B978-0-12-544172-8.00006-2 | isbn = 978-0-12-544172-8 }}</ref> Genes and other genetic information from a wide range of organisms can be inserted into bacteria for storage and modification, creating [[genetically modified bacteria]] in the process. Bacteria are cheap, easy to grow, [[Clone (cell biology)|clonal]], multiply quickly, relatively easy to transform and can be stored at -80&nbsp;°C almost indefinitely. Once a gene is isolated it can be stored inside the bacteria providing an unlimited supply for research.<ref>{{Cite web|url=https://www.learner.org/courses/biology/textbook/gmo/gmo_2.html|title=Rediscovering Biology – Online Textbook: Unit 13 Genetically Modified Organisms|website=www.learner.org|access-date=2017-08-18|archive-date=3 December 2019|archive-url=https://web.archive.org/web/20191203123559/http://www.learner.org/courses/biology/textbook/gmo/gmo_2.html}}</ref>

Organisms are genetically engineered to discover the functions of certain genes. This could be the effect on the phenotype of the organism, where the gene is expressed or what other genes it interacts with. These experiments generally involve loss of function, gain of function, tracking and expression.

* '''Loss of function experiments''', such as in a [[gene knockout]] experiment, in which an organism is engineered to lack the activity of one or more genes. In a simple knockout a copy of the desired gene has been altered to make it non-functional. [[Embryonic stem cells]] incorporate the altered gene, which replaces the already present functional copy. These stem cells are injected into [[blastocyst]]s, which are implanted into surrogate mothers. This allows the experimenter to analyse the defects caused by this [[mutation]] and thereby determine the role of particular genes. It is used especially frequently in [[developmental biology]].<ref name="AlbertsJohnsonLewisRaffRobertsWalter2002">{{Cite book |last1=Alberts |first1=Bruce |last2=Johnson |first2=Alexander |last3=Lewis |first3=Julian |last4=Raff |first4=Martin |last5=Roberts |first5=Keith |last6=Walter |first6=Peter | name-list-style =  vanc |date=2002 |chapter=Studying Gene Expression and Function|url=https://www.ncbi.nlm.nih.gov/books/NBK26818/ |title=Molecular Biology of the Cell |edition=4th |location=New York|publisher=Garland Science |isbn=0-8153-3218-1}}</ref> When this is done by creating a library of genes with point mutations at every position in the area of interest, or even every position in the whole gene, this is called "scanning mutagenesis". The simplest method, and the first to be used, is "alanine scanning", where every position in turn is mutated to the unreactive amino acid [[alanine]].<ref>{{Cite book|url=https://books.google.com/books?id=ycVoWFqDTmAC&pg=PA94|title=Protein Engineering and Design|last1=Park|first1=Sheldon J.|last2=Cochran|first2=Jennifer R.| name-list-style = vanc |date=2009-09-25|publisher=CRC Press|isbn=978-1-4200-7659-2|language=en}}</ref>
* '''Gain of function experiments''', the logical counterpart of knockouts. These are sometimes performed in conjunction with knockout experiments to more finely establish the function of the desired gene. The process is much the same as that in knockout engineering, except that the construct is designed to increase the function of the gene, usually by providing extra copies of the gene or inducing synthesis of the protein more frequently. Gain of function is used to tell whether or not a protein is sufficient for a function, but does not always mean it is required, especially when dealing with genetic or functional redundancy.<ref name="AlbertsJohnsonLewisRaffRobertsWalter2002" />
* '''Tracking experiments''', which seek to gain information about the localisation and interaction of the desired protein. One way to do this is to replace the wild-type gene with a 'fusion' gene, which is a juxtaposition of the wild-type gene with a reporting element such as [[green fluorescent protein]] (GFP) that will allow easy visualisation of the products of the genetic modification. While this is a useful technique, the manipulation can destroy the function of the gene, creating secondary effects and possibly calling into question the results of the experiment. More sophisticated techniques are now in development that can track protein products without mitigating their function, such as the addition of small sequences that will serve as binding motifs to monoclonal antibodies.<ref name="AlbertsJohnsonLewisRaffRobertsWalter2002" />
* '''Expression studies''' aim to discover where and when specific proteins are produced. In these experiments, the DNA sequence before the DNA that codes for a protein, known as a gene's [[promoter (biology)|promoter]], is reintroduced into an organism with the protein coding region replaced by a reporter gene such as GFP or an enzyme that catalyses the production of a dye. Thus the time and place where a particular protein is produced can be observed. Expression studies can be taken a step further by altering the promoter to find which pieces are crucial for the proper expression of the gene and are actually bound by transcription factor proteins; this process is known as [[promoter bashing]].<ref>{{Cite book|url=https://books.google.com/books?id=ed4_CQAAQBAJ&pg=PA114 |title=Techniques in Genetic Engineering|last=Kurnaz|first=Isil Aksan| name-list-style = vanc |date=2015-05-08|publisher=CRC Press|isbn=978-1-4822-6090-8 }}</ref>