Question

There are two stages to producing a knockout mouse. Describe the design and isolation of the construct needed (and the cell type), and then explain the breeding and screening process that occurs to produce the knockout mouse strain.

Answer 1

DESIGN AND ISOLATION: The two method to produce a knockout mouse is by first harvesting embryonic stem cells as they help study the effects even after the gene is knocked out and inserting an artificial DNA into the nuclei of these embryonic cells. This method is called gene targeting. The second method is where an artificial DNA containing a reporter gene is inserted into any gene and not a specific gene of interest.This method is also called as gene trapping. The artificial DNA prevents the process of RNA splicing thereby preventing the expression of a particular protein and intern causing knocking out of its function.

The target vector with target gene like neomycin positive is transfected into the mouse embryonic stem cell by electroporation. The homologous recombinants show a positive selectivity for neomycin where as non homologous show negative selectivity for neomycin. These are tested using southern blotting and PCR. The donor embryonic stem cells are injected into the blastocyst of a mouse. This embryo is the surgically implanted into a pseudo pregnant mouse. Some of the mouse pups born are having both targeted gene while some others do not have target gene. This breeding is continued until a knockout mouse with disruption of target gene in tissues is observed.

BREEDING: After inserting the artificial DNA into the nucleus of embryonic cells they are grown in Petri dishes in the lab and the finally injected into the embryos of mouse. The embryos are then inserted into female mouse uterus and allowed to grow into pups.

SCREENING: some mouse pups will have tissues containing genes that were knocked out while others will have tissues without the knocked out gene. High throughput screening method is used to screen knockout mouse strain. Southernblot and PCR are also used for gene analysis and studying gene expression