Analysis of lymphocyte development and function using the RAG-deficient blastocyst complementation system.
The RAG-deficient blastocyst complementation system (RBCS) represents a flexible and rapid method for the genetic analysis of lymphocyte function using a gene-targeting approach. In chimeras derived from manipulated embryonic stem cells injected into VDJ recombination-incapable, RAG-deficient blastocysts, any lymphoid cells past the prolymphocytic stage will be embryonic stem cell-derived. This approach can therefore bypass pitfalls such as pleiotropy and embryonic lethality to allow the analysis of targeted gene mutations with respect to lymphocyte development and function in a genetically uniform cell population. Thanks to recent advances in targeting techniques and in mouse embryo manipulation, this remarkably efficient technique has become a highly feasible and useful addition to any immunology research program. In this review, we discuss the technical aspects of the procedure, as well as its advantages and drawbacks compared to alternative approaches, and our practical experience in establishing the system at the University of Rochester.
Young
Faith M
FM
Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA.
Pinkert
Carl A
CA
Bottaro
Andrea
A
D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology*; D001755:Blastocyst; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004624:Embryo Transfer; D005260:Female; D005434:Flow Cytometry; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D013234:Stem Cells / Q000166:cytology*; D013601:T-Lymphocytes / Q000166:cytology*; D045567:VDJ Recombinases / Q000235:genetics
D004268:DNA-Binding Proteins; C499308:Rag2 protein, mouse; C064655:V(D)J recombination activating protein 2; D045567:VDJ Recombinases