Authors: S. P. Adhangale, N. S. Chavan
nschavan@kkwagh.edu.in
Gene knockout (GO) is a genetic technique supplemented with biotechnological tool, in which an organism is engineered to carry gene that has been made inoperative. These genes are known as knockouts; used in assigning function to specific genes having unknown function. Gene knockout strategy, reverse genetic tools, used to determine the function of target genes by gene technology, mutagenesis & homologous recombination. Homologous recombination permits gene exchangein vivo. This approach is commonly used in Arabidopsis to nullify the gene function. The technology was primordially developed in yeast Saccharomyces cerevisae. In a study, a group of Melbourne based company & other Japanese Suntory Companies tried to develop world’s biggest blue rose in 2008 by RNAi technology, making a rose with blue pigment. In another study, knockout mouse was first reported through homologous recombination. Knock-out mouse are often used as weapons for studying gene products & their functions.
Introduction
Gene knockout technology is a major component of functional genomics toolbox, comprises a cluster of methods designed to abolish gene function (viz. knockout). Homologous recombination permits gene exchangein vivo. Homologous recombination in plants is only reported in Physcomitrella patens.
Gene knockout technology
An organism in which a single gene of choice or interest is either activated or knocked out in manner that leaves all other genes unaffected by homologous recombination method. The gene knockout is a genetic technique in which an organism is engineered to carry inoperative genes. These organisms carrying such genes are known as knockout organism or simple knock outs. The technique is opposite to gene knock-in. Knocking out of two genes is known as double knockout & similarly the terms triple & quadruple knockouts to describe three or four knockouts genes respectively.
Procedure used for gene knockout technology
- Knockout is accompanied by a combination of techniques beginning in the test tube with plasmid, bacterial artificial chromosome (BAC’s) or other DNA construct.
- The goal is to create transgenic animals with an altered gene. For this, embryonic stem cells are genetically transformed & inserted into early embryos.
- Resulting animals with genetic change in germ lines can pass gene knockout to future generation
The expression of inserting a T-DNA element into the genome depends on the nature of T-DNA & location of insertion point.
The “knock-out” mutation includes insertion of T-DNA in coding or promoter region, as a result gene expression completely disrupts. The knock-down mutation includes insertion of T-DNA in promoter or 3 1 untranslated regions. The knock-on mutation are typical mutations in which DNA construct carries a constitutive promoter, such as cauliflower mosaic virus 35 S promoter. The knock-about mutation includes insertion of T-DNA in promoter or coding region. The knock-worst mutation T-DNA insertion resulting in chromosomal rearrangements. E.g. Arabidopsis
Arabidopsis g ene knockout technology: Phenotypic discovery
The Arabidopsis Genome Initiative (TAGI) was started genome sequencing of Columbia strain of Arabidopsis thaliana (1996) by International Collaboration in Dec.2000. PCR primers specific for both the target genes & insertion element are used.
Characterization of knockout phenotypes
Analysis of knockout lines in Arabidopsis however showed that DAG1 is involved in control of seed dormancy. DAG1 plants have distorted siliqua with seeds that germinate earlier than normal, do not require light for germination. Plants are sessile organism have evolved many adaptive traits that allow them to cope with biotic & abiotic traits.
Creation of world’s first blue rose
The CSIRO developed RNAi technology under license by Florigene Ltd., a Melbourne based Biotechnology Company & a part of Japanese Suntory Companies (2008)
There are three steps:
- Turn off the production of red pigment- Check the rose DFR (dihydroflavnol reductase) gene making red pigment by silencing.
- Open the door to production of blue pigments- Opened by gene insertion (viz.delphividin) gene from pansy. This opened the door for production o blue pigment in rose flower
- Produce blue pigment- Replace the door DFR gene from an Iris, excellent in producing blue pigment Iris DFR gene was inserted in rose & subsequently rose with blue flower was produced Rose petals are moderately acidic with pH 4.5 Blue flowers should be achievable if rose petals can be made less acidic; as acidity inhibits blue pigments
- Blue rose have been available in Australia since 1996.
- They allow testing specific function of particular gene & observing their process of regulation.
- Prions related proteins (PRP’s) is another knockout technology in animals including inactivation of PRP genes
E.g. Bovine Spongiform Encephalopathy (BSE), Scrapia etc.
Drawbacks of gene knockout
- About 15% of gene knockout developed are lethal
- Knocking out of genes may also fail to produce an observable change (e.g. in mouse)
- Deletion of genes could eliminate a major target of hyper acute rejection response & thus lead to the development of animals serving as human donors.
- Knocking out is activity of genes to provide information about what the gene normally does.
- Its development has been a massive advance to biomedical & pharmaceutical field.
- Knockout mouse is a wonderful tool used to study function of specific gene.
- The Florigene blue rose is doubly historic & first rose in the world having potential to produce ‘true blue’ roses
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