Treatment of genetic immune deficiency in the mouse by therapeutic cloning.

The Rag2^−/− mouse lacks all B and T cell function as a result of homozygous deletion of the Recombinase Activating Gene 2 (Rag2). Cells from a clipping of the mouse tail were cultured briefly. Using micromanipulation, the nucleus of a tail-tip cell was removed and inserted into a donor egg, whose own nucleus had been removed by micromanipulation. The reconstructed zygote underwent cleavage and development to blastocyst stage, after which the cells of the inner cell mass were removed and placed in culture, forming an ES cell line with genetic equivalence to the Rag2^−/− mouse. One of the two defective Rag2 alleles was replaced with an intact copy by homologous recombination, generating a repaired ntES^Rag2+/− cell line. These cells were differentiated in vitro into hematopoietic stem cells, and used to transplant irradiated immune-deficient Rag2^−/− recipients. Engrafted mice showed restoration of T and B cell populations and production of serum immunoglobulin, demonstrating the feasibility of combined gene and cell therapy (therapeutic cloning; Rideout et al²⁷).