KNOCKED OUT AN EXISTING GENE BY REPLACING IT OR DISRUPTING IT WITH AN ARTIFICIAL PIECE OF DNA.

KNOCKOUTS ARE USED TO STUDY THE FUNCTION OF SPECIFIC GENES.

THE FIRST RECORDED KNOCKOUT MOUSE WAS CREATED BY MARIO R. CAPECCHI, MARTIN EVANS AND OLIVER SMITHIES IN 1989

GENE KNOCK OUT TECHNOLOGY

A laboratory mouse in which a gene affecting hair growth has been knocked out (left), is shown next

to a normal lab mouse

KNOCKOUT MICE ARE A COMMON TOOL FOR STUDYING GENES IN THE LABORATORY.

MICROORGANISMS ARE ALSO USED FOR STUDYING GENE FUNCTION.

THE KNOCKOUT CAN BE LETHAL.

ANIMAL MODELS

WITH THE USE OF RECOMBINANT DNA TECHNOLOGY CLONED GENES ARE MODIFIED IN SUCH A WAY, THAT THEY ARE NO LONGER FUNCTIONAL, CLONED GENES MODIFIED IN THIS WAY ARE KNOWN AS ‘KNOCK OUT

CONSTRUCT’, WHEN HOMOLOGOUS RECOMBINATION OCCURS WITH A KNOCK OUT CONSTRUCT.THE NON-FUNCTIONAL KNOCKOUT ALLELE

REPLACE THE ENDOGENOUS MILD TYPE ALLELE.

HOW IT WORKS?

THE GENE TO BE KNOCKED OUT IS ISOLATED FROM A MOUSE GENE LIBRARY

THEN A NEW DNA SEQUENCE IS ENGINEERED

THE NEW SEQUENCE IS ALSO GIVEN A MARKER GENE.

STEM CELLS ARE ISOLATED FROM A MOUSE BLASTOCYST.

PROCEDURE

,

• The new sequence is introduced into the stem cells by electroporation.

The stem cells that incorporated the knocked-out gene are isolated.

The knocked-out stem cells are inserted into a mouse blastocyst.

These blastocysts are then implanted into the uterus of female mice

• The newborn mice will therefore be chimeras.

These chimera mice are crossbred with others of the wild type.

The mice produced are not chimeras, but they are still heterozygous.

When these heterozygous offspring are interbred, some of their offspring will inherit

the knocked-out gene from both parents;

KNOCKOUT MOUSE GENE IN ES CELLS

APPLICATIONS

To determine the function of gene products.To create mouse modal of human genetic diseases.To characterize genetic regulatory regions.

CYSTIC FIBROSISCystic fibrosis is a genetic disorder that perticularly

affects the lungs and diagestive system.CF is inherited disease.The inherited CF gene directs the body’s epithelial

cells to produce a defective form of a protein called CFTR(cystic fibrosis transmembrane conductance regulator) found in cell line of lungs,digestive tracts,sweat gland,and genitourinary system.

CF is inherited disease.The inherited CF gene directs the body’s epithelial cells to

produce a defective form of a protein called CFTR(cystic fibrosis transmembrane conductance regulator) found in cell line of lungs,digestive tracts,sweat gland,and genitourinary system.

When the CFTR protein is defective,epithelial cells can’t regulate the way chloride (part of salt called sodium chloride) passes across cell membrane.

• This disrupts the essential balance of salt and water needed to maintain a normal thin coating of fluid and

mucus insight the lungs, pancreas and passege ways in other organs.The mucus become thick, sticky and hard

to move.Normally, mucus in lungs traps germs,which are then

cleared out of the lung. But, in CF, the thick,sticky mucus and germs it has trapped remain in lungs, which

become infected.

• The human Cystic Fibrosis gene is identified and cloned in a phage vector.ES cells are recoverd from mouse blastocyst.Transfection of ES cells with CFTR knockout construct.Animals homozygous for the CFTR knock out allele display a mutant phenotype that is very similar to that expressed by humans suffering from Cystic Fibrosis.Thus ,in developing drugs to alleviate CF system in human,pharmaceutical researches can first test new products in mice to determine their efficiency.

LIMITATIONS

Genetically altered embryos cannot grow into adult mice.

The lack of adult mice limits studies to embryonic development.

The gene may serve a different function in adults than in developing embryos.