Mammalian Developmental Genetics 
the molecular pathways of mammalian segmenta- 
tion and the role of Hox-2 genes in rhombomere 
morphogenesis. 
Insertional Mutagenesis 
An important development in mammalian ex- 
perimental embryology has been the ability to 
isolate embryonic stem cells from preimplanta- 
tion mouse embry^os, 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 /3-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 eff^ects. To block this expression in a single 
step, and in a conditional fashion, we have con- 
structed a series of gene trap vectors that contain 
an inducible promoter on the strand opposite 
the reporter sequences. "Captured" cell clones 
are first selected by antibiotic resistance or /3- 
galactosidase staining. After removal of selective 
pressure and activation of the inducible pro- 
moter, an antisense transcript is generated against 
coding sequences of the endogenous gene. In 
certain cases, this 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 eff'ects 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. 
Ultimately we plan to place the gene trap vec- 
tors in the context of retroviral packaging ele- 
ments to allow their introduction into the mouse 
germline by infection of developing gametes. 
Theoretical advantages of using retrotransposons 
as insertional mutagens include an enzymatic 
mechanism of integration that conserves host 
DNA sequences, accessibility to most if not all 
segments of the genome, and the efficient detec- 
tion and recovery of new integration sites. These 
advantages, however, have been difficult to 
achieve in the mouse, in part because of the low 
efficiency of retrotransposition in the germline. 
In the case of endogenous ecotropic proviruses 
and exogenous Moloney murine leukemia virus 
(MoMuLV) , a block to retroviral infection of de- 
veloping gametes seems likely to be due to a com- 
bination of factors, including decreased accessi- 
bility of germline cells to viral particles with very 
short infectious half-lives and a transcriptional 
block to expression from the retroviral long ter- 
minal repeat (LTR). As a first step toward over- 
coming these hurdles, we have constructed, in 
collaboration with Patrick Brown (HHMI, Stan- 
ford University) , transgenic mice that contain the 
transcriptional control sequences for the mouse 
protamine 1 gene fused to the coding sequences 
for MoMuLV. Because the mouse protamine 1 
gene is highly expressed in postmeiotic sperma- 
tids, the transgene should provide a rich source 
of native retroviral particles in the vicinity of de- 
veloping male gametes. Our preliminary results 
show that the chimeric protamine-MoMuLV 
transgene is functional and can lead to retroviral 
infection of somatic and germline tissues. 
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