Knock-out mouse

A knock-out mouse is a transgenic tool used to study the phenotype of a mouse when a gene is gknocked-outh or replaced with a gene knock-out.  A gene knock-out is a vector containing a segment of manipulated DNA that does not produce a viable product.  This vector is created with flanking regions that are homologous to the area of the chromosome where one wishes the manipulated gene to integrate. When this vector is introduced into the embryonic stem cell of a mouse it undergoes replication as the embryonic stem cell divides.  In a small number of the divided cells it replaces the original gene segment through a process known as homologous recombination.  A good animation of homologous recombination is at Hannam Universityfs microbiology website:

http://genetics.hannam.ac.kr/lecture/rec-1.htm

 

Within the introduced vector is a marker, usually a sequence that encodes for a protein.  This protein will enable the host cell to be resistant to some sort of drug.  Through this process the cells, in which the vector was integrated and those in which it was not, are screened.  A good illustration of this screening process is shown in the figure below.

 

As seen, the replacement vector has been introduced and through homologous recombination, the vector has been integrated into the chromosome.  A sequence for resistance to neomycin has been placed in the vector, allowing for positive selection.  In order to make sure of homologous recombination, opposed to random insertion, gancyclovir is introduced.  With the introduction of gancyclovir, negative selection screens the rest of the cells with random insertion.  This is due to the original gene sequence`s sensivity to gancyclovir . 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

After it has been found that the knock out gene has been integrated in the appropiate place, the stem cells are then introduced into another embryo.  This is where the breeding of a knock-out line begins.  A good illustration and explanation of the breeding process was available at the Fred Hutchinson Cancer Research Center web page and is shown below:

 


 

 

Thus, at the end of the breeding line, the knock-out gene is expressed. The mouse shows, in the complete absence of a viable copy of the gene, what function the gene had. 

 

Knock-in mouse

 

A knock-in mouse is similar to a knock-out mouse in the process by which the gene is introduced and the mechanisms in order to check for homologous recombination of the gene.  However, with a knock-in mouse, a specific allele, or locus or base sequence is manipulated in order to observe specific functions within the gene.  An example pointed out by Dr. Evans of the Clinical Cancer Genetics Services at the University of North Carolina Hospital is delta f 508, the most common mutation in cystic fibrosis.  Within knock-in mice, this allele is usually over expressed in order to study its effects upon the morphology and physiology of mice.  Thus, knock-in mice are more specific for mutations of specific alleles or locus within the gene. Also, knock-in mice produce a viable genetic product such as an altered protein. Viability and specificity within knock-in mice are the points of contrast to knock-out mice.

 

Since it`s inception some decades ago knock-out mice have enabled scientist to understand the function of a gene in the absence of its function.  However, as genetic engineering has progressed scientist are now able to make more precise manipulations of a single allele within a gene.  Thus, specific functions of the gene, opposed to only the functional absence, can be studied using knock-in mice.

 

I would like to thank Dr Jim Evans of  The Clinical Cancer Genetics Services at the University of North Carolina Hospital and Dr. David Threadgill of Lineberger Comprehensive Cancer Center for their help in answering specific questions. 

 

I would also like to thank MITA for allowing me to undertake this small task, and Professor Barron for not accepting my answer on mere faith alone. 

 

If you are interested in knowing more specifics about knock out and knock-in mice please check out the following links:

 

www.webmd.com

This is a great website that gives a very general overview through its reference sources, as well as a very specific analysis through its journal library.  Only downfall is that you need a membership.  However, you can apply for a 10 day free membership at the website.

http://meds.queensu.ca/medicine/crl/greer/kno_pag.htm

If my explanation was not good enough, this lab gives a short discussion as to what knock-out and knock-in mice are and how they are made.

http://genetics.hannam.ac.kr/lecture/Mgen02/Recombination.htm

Hannam Universityfs Microbiology Dept., this website offers a lot of good animation clips concerning the differing types of recombination.

http://www.genoway.com/service.htm

A pharmaceutical company that explains the advantages of knock-in mice over other transgenic methods, and also lists some specific knock-in mutations they have readily available