Eugeniu Nacu

GENE KNOCKOUT BY TARGETED DISRUPTION

Principle: by targeted disruption is used to generate transgenic mice containing a defective copy of a particular gene. By comparing the wild-type organism with the mutant mouse the role of the respective gene can be elucidated.

Objective: - Generate a DNA construct containing a mutant of the gene of interest - Generate knock-out mice - deficient in the protein of interest - Study the role of the protein by looking at the effect caused by absence

Procedure:

A DNA construct containing the gene of interest and the thymidine kinase gene (HSV-tk) is generated. The gene of interest is interrupted by inserting an antibiotic resistance marker. The DNA is introduced into the embryonic stem cells with three possible outcomes: a) The gene from the DNA construct undergoes homologous recombination with the endogenous copy of the gene thus disrupting it and resulting in the insertion of the neomycin-resistance gene into the genome. The HSV-tk gene will be left on the DNA construct and will be eventually lost from the cells. This will bestow resistance to neomycin-like drugs and ganciclovir, respectively, upon the cells. b) The DNA construct fails to integrate and will be lost. Thus the cells will Fig 1. A hypothetical DNA construct and the possible outcomes lack neomycin resistance gene and of its insertion into cells (Janeway et al., 2001) will be killed upon G418 (neomycin- like drug) treatment c) The DNA construct incorporates randomly into the ’s genome. These cells will express both neomycin-ressitance and HSV-tk. The presence of HSV-tk causes the cells to be susceptible to ganciclovir and thus to be killed upon treatment with it.

Using G418 and ganciclovir it is possible to select only those embryonic stem cells which have undergone homologous recombination. In order to achieve homozygous disruption of the both alleles in diploid cells two different constructs, disrupted by one of the two different markers, are used. In order to study the absence of the gene in vivo it is sufficient to generate heterozygous embryonic stem cells: Eugeniu Nacu - The embryonic stem cells heterozygous for the mutant gene are inserted into a blastocyst which is reimplanted in the uterus of a surrogate pregnant mouse. The implanted cells give rise to different types of cells in the offspring, thus generating chimaeras. - Hopefully some of the chimeric offspring contain a heterozygous germ line. These chimeric mice are interbred to hopefully obtain mice heterozygous for the mutation. - The heterozygous mice are interbred until mice homozygous for the mutation are born. These homozygous mutant mice are completely deficient in the protein under investigation and are used to study the effect of the knock-out and thus the function of the protein. As a control - if a particular phenotype is reversed upon introduction of a functional copy of the investigated gene into the cells/animals then it is possible to conclude that certain effects/phenotypes are a direct result of the absence of the protein,.

A problem arises when the gene of interest is a recessive lethal gene, meaning – the mice homozygous for the absence/mutation of the gene die shortly post-birth or the embryo itself doesn’t develop. However, it is possible to study the function of the lethal gene in lymphoid cells by making chimeras of mice deficient in B and T cells: - Embryonic stem cells homozygous for the lethal mutation are inserted into the blastocyst from mice lacking the recombinase-activating genes (RAG) which are thus unable to rearrange their antigen-receptor genes. - As the embryo develops the RAG deficient cells compensate for the developmental deficiencies due to the homozygous cells. - The RAG deficient cells can’t develop the lymphoid system, which in turn arises from the homozygous mutant embryonic stem cells. Thus the chimeric mice that are born contain the knock-out of the gene only in the cells of the lymphoid system.

Example:

In Fig 2 is shown how the vectors, to be used for homologous recombination, were designed, and the outcome of the recombination is also indicated. The targeting vector (top) has the exons, from 4 till 8 of the wild type ERAAP (ER aminopeptidase associated with antigen Fig 2. The construct used for the generation of knock-out mice (Figure 1a processing) gene taken from Shastri et al., Nature Immunology 7 (2006). P. 103-112) (middle), removed and substituted by the gene conferring neomycin resistance. Although not indicated in the figure the construct (top) contains a gene coding for the diphtheria toxin allowing for negative selection of cells in which random integration of the construct into the genome has occurred. The product of homologous recombination between the targeting vector/construct and the endogenous wild type gene is shown in the diagram labeled mutant (ko), at the bottom of the figure.

References: Janeway CA, Travers P, Walport M, Shlomchik M, (2001). Immunobiology 5 : the immune system in health and disease. Garland Publishing. NY.Hammer GE, Gonzalez F, Champsaur M, Cado D & Shastri N (2006). The aminopeptidase, ERAAP, shapes the peptide repertoire displayed by major histocompatibility complex class I molecules. Nature Immunology. 7:1:103-112.