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283 
to achieve, and in particular, electroporation protocols established for 
mouse ES cells do not v\^ork in human ES cells [22]. Also, in contrast 
to mouse ES cells, human ES cells proliferate inefficiently from single 
cells, making screening procedures to identify rare homologous 
recombination events difficult [12]. We have recently developed 
modified electroporation protocols to overcome these problems and 
have successfully targeted a ubiquitously expressed gene {HPRTl), 
an ES cell-specific gene {POU5F1), and a tissue-specific gene 
(Tyrosine hydroxylase: TH) in human ES cells [22, 44]. The overall 
targeting frequencies for the three genes suggest that homologous 
recombination is a broadly applicable technique in human ES cells. 
Homologous recombination in human ES cells will be 
important for studying gene function in vitro and for lineage 
selection. For therapeutic applications in transplantation medicine, 
controlled modification of specific genes should be useful for 
purifying specific ES cell-derived, differentiated cell types firom a 
mixed population [45]; altering the antigenicity of ES cell derivatives; 
and giving cells new properties (such as viral resistance) to combat 
specific diseases. Homologous recombination in human ES cells 
might also be used for approaches combining therapeutic cloning 
with gene therapy [46]. In vitro studies using homologous 
recombination in human ES cells will be particularly useful for 
learning more about the pathogenesis of diseases where mouse 
models have proven inadequate. For example, HPRT-deficient mice 
fail to demonstrate an abnormal phenotype, yet defects in this gene 
cause Lesch-Nyhan disease in children [47]. In vitro neural 
differentiation of HPRT-deficient human ES cells or transplantation of 
ES cell-derived neural tissue to an animal model could help to 
understand the pathogenesis of Lesch-Nyhan syndrome. 
Human ES Cells as a Model of Early Human Development 
The excitement surrounding the prospective role of human 
embryonic stem (ES) cells in transplantation therapy has often 
overshadowed a potentially more important role as a basic research 
tool for understanding the development and function of human 
tissues. The use of human ES cells is particularly valuable to derive 
tissue for study that is difficult to obtain otherwise, and for which 
animal models are inadequate. 
Human ES cells offer a new and unique window into the 
early events of human development, a period critical for 
understanding infertility, birth defects, and misccirriage. Because 
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