Developmental Genetics in the Mouse and Human 
Conditional Inactivation of Recessive Genes 
in Embryonic Stem Cells 
An important development in mammalian ex- 
perimental embryology has been the ability to 
isolate embryonic stem cells from preimplanta- 
tion mouse embryos, which can be modified in 
cell culture and then used to reconstitute an in- 
tact animal. Like the whole organism, these cells 
are diploid and contain two copies of every auto- 
somal gene. When embryonic stem cells are 
mixed with a fragment of "reporter" DNA, such 
as the coding regions of the neomycin resistance 
gene or the ^-galactosidase gene, insertion of the 
exogenous DNA provides a "gene trap," in which 
expression of the reporter sequences is con- 
trolled by regulatory elements of an endogenous 
gene. Insertion of the reporter DNA is likely to 
disrupt expression of the endogenous gene, but 
in most cases, expression from the uninterrupted 
homologue will be sufficient to prevent pheno- 
typic effects. 
To block this expression in a single step, and in 
a conditional fashion, we have constructed a se- 
ries of gene trap vectors that contain an inducible 
promoter on the strand opposite the reporter se- 
quences. "Captured" cell clones are first se- 
lected by antibiotic resistance or |8-galactosidase 
staining. After removal of selective pressure and 
activation of the inducible promoter, an anti- 
sense transcript is generated against coding se- 
quences of the endogenous gene. In certain 
cases, this antisense transcript will function in 
trans, thereby inhibiting expression of the endog- 
enous gene from the uninterrupted homologue. 
Control experiments suggest that these DNA 
vectors are capable of trapping endogenous 
genes and that basal expression of the inducible 
promoter does not reduce appreciably the fre- 
quency of trapping. We are currently isolating a 
panel of captured cell clones. The efficiency of 
these antisense promoters in cis will be analyzed 
by measuring expression of the reporter se- 
quences before and after antisense induction. Po- 
tential effects of trans inhibition will then be 
tested by examining the phenotypes of chimeric 
mice that contain the mutant embryonic stem 
cells. This approach will allow the phenotypic 
effects of a recessive mutation to be studied in a 
diploid organism by altering only one of the two 
copies, and may be applicable to many organisms 
and developmental systems. 
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