knockout mice

                                         

                                      knockout mice

Knock out mice is a mice in which particular gene e.g. [IL-2 gene] is replaced by muted version of the cell. The first knock out mice was created by Mario martin and Oliver in 1989 for this received Nobel prize in 2007 knockout mice are general used to study function of particular gene and to study immune deficiency disorder.

Procedure to create knock out mice

Part A: -Constructed of mutated gene

Gene to be modified. Is selected and isolated. Offer this similar copies in engineered and constructed with few modifications generally marker genes are in sorted for modification. These are two types of marker gene. Neomycin resistance gene with confirm resistance to neomycin is inserted inside the target gene and tyrosine kinase gene of harpies’ simplex virus with confirms susceptibility to glancycloveridity in inserted outside the target gene.

Part B: -Collection of embryonic stem cell`s

 Blastocyst is surgically isolated from the white mouse in the inner site of blastocyst there is a bunch of cells which known as inner mass. This cell mass is composed of embryonic stem cell. This embryonic stem cell are isolated and cultured I artificial media.

Part C: -Insertion of modified gene

Ø The modified gene is transfected in the embryonic stem cell by electroporation.

Ø  due to electroporation cell membrane of embryonic stem cell become pores eana DNA fragment is transferred inside.

Ø  The probability of transfection is very low generally 1%.

Ø If the DNA is transferred inside the cell. There are 2 probability in which the DNA is inserted in the chromosome of cell i.e. either homologous recombination or by heterologous recombination.

Ø  If homologous recombination mutant gene will undergo recombination with normal copy of gene present In chromosome. In this case mutated gene containing neomycin resistant gene will be tyrosine kinase gene will be detected.

Ø If heterorecombination occur the complete modified can be inserted anywhere In the chromosome it will contain both neomycin resistance and tyrosine kinase gene the cell`s which have undergone homologous recombination are generally heterozygous [i.e. only 1 copies of is replace by mutated gene while other remines unaffected.

Part D: -Selected of heterozygous cell

Ø For selection the cells are transferred on the media, containing neomycin and gancycloveridity.

Ø  normal cells [which are not undergone] homologous heterologous recombinant. Lack of neomycin resistance gene and hence will be killed. By neomycin.

Ø  cells which have undergone heterologous recombinant (which contain neomycin resistance and tyrosine kinase gene will be killed by glancycloveridity.

Ø cells which have undergone homologous resistance gene will survive in this media because they are resistance to both neomycin and glancycloveridity.

Part E: -Implantation of modified embryonic  

Ø The modified cells [heterozygous for target gene] are transferred to blastocyst from gray mice and this blastocyst is then planted to the uterus of faster mother or pseudo pregnant mother.

Ø This mother uses give birth to two types of progeny. Chimeric progeny (produced from two different embryonic stem cells) and grey progeny.

Ø Then progeny is then crossed with grey progeny this will give birth to some white progeny which are heterozygous for target gene.

Ø This progeny is again interbred which will finally give birth to homozygous. White progeny which have both the copies of target gene mutated.

Ø In this way both the copies of target gene are made non-functional.

Application of knock out mice

Ø Knockout mice have been useful include studying and modeling different kinds of cancer, heart disease, arthritis substance, abuse, aging and perkiness disease.

Ø Knockout mice also offer a biological and scientific contact in which and other therapies can be developed and tasted.