On this page, we provide several useful reference papers for your review.

1.Manipulation of Mouse Embryonic Stem Cells for Knockout Mouse Production
Advait Limaye, Bradford Hall, and Ashok B. Kulkarni

Abstract: The establishment of mouse embryonic stem (ES) cell lines has allowed for the gene?ration of the knockout mouse. ES cells that are genetically altered in culture can then be manipulated to derive a whole mouse containing the desired mutation. To successfully generate a knockout mouse, however, the ES cells must be carefully cultivated in a pluripotent state throughout the gene-targeting experiment. This unit describes detailed step-by-step protocols, reagents, equipment, and strategies needed for the successful generation of gene knockout embryonic stem cells using homologous recombination technologies. Curr. Protoc. Cell Biol. 44:19.13.1-19.13.24.

2. Gene Knockout Protocols: Second Edition
Editor(s): Wolfgang Wurst, Ralf Kühn
Series: Methods in Molecular Biologg. Volume No.: 530

3. Gene targeting: a practical approach.  A Book Edited By Alexandra L. Joyner.

4. Wu S , Ying G , Wu Q , Capecchi MR (2008). A protocol for constructing gene targeting vectors: generating knockout mice for the cadherin family and beyond. Nat Protoc. 3(6): 1056

We describe here a streamlined procedure for targeting vector construction, which often is a limiting factor for gene targeting (knockout) technology. This procedure combines various highly efficient recombination-based cloning methods in bacteria, consisting of three steps. First step is the use of Red-pathway-mediated recombination (recombineering) to capture a genomic fragment into a Gateway-compatible vector. Second, the vector is modified by recombineering to include a positive selection gene neo, from a variety of modular reagents. Finally, through a simple in vitro Gateway recombination, the modified genomic fragment is switched into a vector that contains negative selection cassettes, as well as unique sites for linearization. To demonstrate the usefulness of this protocol, we report targeted disruptions of members of the cadherin gene family, focusing on those that have not been previously studied at the molecular genetic level. This protocol needs 2 weeks to construct a targeting vector, and several vectors can be easily handled simultaneously using common laboratory setup.