GENE KNOCKOUT

A gene knockout is a genetic technique in which one of an organism's genes is made inoperative ("knocked out" of the organism). However, gene knockout can also refer to the gene that is knocked out or the organism that carries the gene knockout. Knockout organisms or simply knockouts are used to study gene function, usually by investigating the effect of gene loss. Researchers draw inferences from the difference between the knockout organism and normal individuals.

1. GENE KNOCKOUT

2. ABSTRACT A gene knockout is a genetic technique in which one of an organism's genes is made inoperative ("knocked out" of the organism). However, gene knockout can also refer to the gene that is knocked out or the organism that carries the gene knockout. Knockout organisms or simply knockouts are used to study gene function, usually by investigating the effect of gene loss. Researchers draw inferences from the difference between the knockout organism and normal individuals.

3. GENE KNOCKOUT A gene knockout (abbreviation: KO) is a genetic technique in which one of an organism's genes is made inoperative ("knocked out" of the organism). However, KO can also refer to the gene that is knocked out or the organism that carries the gene knockout. Knockout organisms or simply knockouts are used to study gene function, usually by investigating the effect of gene loss. Researchers draw inferences from the difference between the knockout organism and normal individuals. The KO technique is essentially the opposite of a gene knockin . Knocking out two genes simultaneously in an organism is known as a double knockout (DKO). Similarly the terms triple knockout (TKO) and quadruple knockouts (QKO) are used to describe three or four knocked out genes, respectively.

4. Technologies for gene-knockout The best approach to produce a gene knockout is homologous recombination and through gene knockout methods a single gene gets deleted without effecting the all other genes in an organism. With the help of the gene knockout the organism where the gene of interest becomes inoperative is known as knockout organism. When more than one gene is get knocked out in an organism then is called double knock out or DKO, triple knockout or TKO and quadrule knockouts or QKO depending on the number of genes.

5. Gene technology procedure Gene knockout is carried out together with elements such as plasmid, DNA construct or bacterial artificial chromosome. Gene knock out procedure often generate transgene animals where the target gene has been altered. To produce transgenic animals, embryonic stem cells or ES cells get genetically modified and in following step the transformed ES cells are placed in early embryos. The transformed animals thus produce has the ability to carry forward the transformed gene in following generations

6. Equipment Thermo cycler for PCR Agarose gel apparatus Power supply Spectrophotometer to read DNA concentration at 260nm and cell density at 600nm Electroporator Constant temperature bacterial incubator set at 30°-32°C. Should contain a roller for liquid culture tubes and shelves for petri plates 32° and 42°C shaking (200rpm) water baths (42°C cannot be an air shaker) Low-speed centrifuge with Sorvall SA-600 rotor (or equivalent) at 4°C Refrigerated microcentrifuge at 4°C Gel imaging system Insulated ice bucket Sterile 35 to 50 ml plastic centrifuge tubes Sterile 50 and 125 ml (or 250) Erlenmeyer flasks, preferably baffled Micropipettors Sterile, aerosol-resistant pipettor tips Pipettes of various sizes PCR tubes (0.2 ml flat cap tubes) 5 ml microfuge tubes Sterile glass culture tubes with stainless steel closures for culturing bacteria Spectrophotometer cuvettes Electrotransformation cuvettes with 0.1cm gap (pre-chilled) Petri Plates – 100 x 15mm Optional but highly recommended: DNA analysis software

7. Different Methods for gene knockout Homologous Recombination- homologous recombination is the conventional method for gene knockout and widely used in genome engineering. This method comprises of nucleotide exchange between DNA sequences which are either similar or identical. Homologous recombination method includes a DNA construct with the mutation of choice and a drug resistance cassette to be interchanged in place of knockout gene. Additionally, the construct also includes a homologous region of nearly 2Kb with the target gene. Microinjection or electroporation are the most common methods which are next applied to transfer the construct into desired organism.

8. Homologous recombination Traditionally, homologous recombination was the main method for causing a gene knockout. This method involves creating a DNA construct containing the desired mutation. For knockout purposes, this typically involves a drug resistance marker in place of the desired knockout gene. The construct will also contain a minimum of 2kb of homology to the target sequence. The construct can be delivered to stem cells either through microinjection or electroporation. This method then relies on the cell's own repair mechanisms to recombine the DNA construct into the existing DNA. This results in the sequence of the gene being altered, and most cases the gene will be translated into a nonfunctional protein, if it is translated at all.

9. Contd. Site-Specific Nucleases- There are namely three methods, zinc fingers, TALENS and CRISPER which is known to introduce double stranded breaks in DNA. Following DNA damage, the cells own repair mechanism get functional through non-homologous end joining (NHEJ), to ligate two open ends. The repair mechanism being finished imperfectly generates insertion or deletion mutation which results in frame shift mutation. Following the mutation the gene produces non-functional protein and generates a knockout for the gene of interest.

10. Zinc-Fingers Applications- Zinc finger nucleases (ZFNs) are used for creating complete knockout in several cell lines. They can be used in cell based screening methods by generating knock-in cells lines where endogenous target genes can be tagged with promoter, reporter or fusion tag proteins. They are also useful for generating cell lines to produce specific protein or antibodies in higher amount.

11. TALENS Applications- TALENs are used widely for plant genome modification . They are useful for the production of biofuels. They have been used to generate knockout in organisms such as zebrafish, rat , mice or c.elegance It also been used to treat genetic diseases such as xeroderma pigmentation ot sickle cell .

12. CRISPER/Cas9 Applications- CRISPER/Cas9 gene editing system is useful for Homology-directed repair (HDR) mechanism. They can be used for gene silencing They are functional for transiently activate endogenous genes. It is useful for DNA free gene editing methods. They are used for transiently silenced expression of genes. Preparation of transgenic animals as well as embryonic stem cells.

13. USES Knockouts are primarily used to understand the role of a specific gene or DNA region by comparing the knockout organism to a wildtype with a similar genetic background. Knockout organisms are also used as screening tools in the development of drugs, to target specific biological processes or deficiencies by using a specific knockout, or to understand the mechanism of action of a drug by using a library of knockout organisms spanning the entire genome, such as in Saccharomyces cerevisiae.

14. REFERENCE National Programme for Technology Enhanced Learning, a project funded by MHRD, Govt. of India. Molecular Biology Of The Gene by James. D. Watson, PEARSON Publication. https://en.wikipedia.org/wiki/Gene_knockout Biotechnology by P.K.Gupta , Rastogi Publication. Biotechnology by B.D.Singh , Kalpana publication.