During the last two decades, cultured epithelial autografts (CEAs) have been used for the treatment of full thickness injury and can be life saving for individuals having sustained injuries covering >80% total body surface area. However, this therapeutic option suffers in two ways: first, CEA production for coverage of large body surface areas requires 17-21days and second, during the initial engraftment period the epithelium is quite fragile. To address the first issue, alternative methods have been reported utilizing cells harvested prior to confluence and therefore requiring less time for in vitro expansion. These methods supply cells either as a pre-confluent, but transportable, epithelial sheets grown on fibrin glue or as suspensions to be sprayed onto wounds within a fibrin sealant. In both cases growth to confluence is achieved in situ. The comparative usefulness and fragility of these modified products await clinical trials. However, in the event of mass casualties with extensive (>80% TBSA) full thickness burn injuries, the usefulness of CEAs or cell sprays may be limited, as the infrastructure does not exist to produce these “skin replacements” for many hundreds or thousands of individuals simultaneously. We have been developing a genetically modified keratinocyte strain that may be used to address this particular problem. The strain chosen has extensive in vitro proliferative potential. The genetic alterations, which will be introduced into the strain, include those able to reduce immune competence and thereby to generate a “universal” keratinocyte donor. As a first step, we are suppressing the expression of both MHC class I and class II so that the keratinocyte will no longer serve as a non-professional antigen presenting cell. We have identified specific artificial RNAs, antisense and siRNA, for β2-microglobulin and the MHC class II invariant chain (Ii) and tested their function in Raji cells. Of the 4 antisense libraries tested and the 4 siRNAs evaluated, we were able to identify defined antisense RNA and siRNA capable of suppressing the expression of MHC class I and II, respectively. As “Raji” cells express higher levels of both MHC class I and class II than do γ-interferon (IFNγ) stimulated keratinocytes, the results predict that keratinocytes harboring both artificial RNAs will not express MHC class I or class II even after stimulation. The immuno-stimulatory capabilities of the genetically modified keratinocytes (MHC class I/II-/-) will be tested first in vitro using FACS analyses, ability to sensitize T cells in mixed lymphocyte reaction and effectors functions in a chromium release cytotoxicity assay. If the experimental results are positive, these cells will be grafted onto humanized SCID mice for evaluation of transplant stability. The necessity for introducing additional genes whose products are known to limit the development of inflammatory and immune responses will be experimentally determined.