DEVELOPMENTAL DISORDERS IN TRANSGENIC MICE 
Paul A. Overbeek, Ph.D., Assistant Investigator 
Research over the past year has made use of 
transgenic mice to study mammalian development. 
Two of the transgenic families under study {down- 
less and spermless) have developmental abnormali- 
ties caused by insertional mutations. For both of 
these families, genomic sequences flanking the trans- 
genic inserts have been cloned and used to search 
for the coding sequences of the inactivated genes. 
In addition to the insertional mutants, the a-crys- 
tallin promoter has been used to direct lens-specific 
expression in transgenic mice. Two different con- 
structs have been studied in some detail: one en- 
codes a truncated SV40 early region, the other 
codes for transforming growth factor a (TGF-a). 
L downless. 
In one transgenic family, integration of the trans- 
genic DNA inactivated the downless gene. This 
gene had been identified previously by classical ge- 
netics and had been mapped to mouse chromo- 
some 10. Single-copy genomic sequences flanking 
the site of integration in the transgenic family were 
cloned and shown to map to mouse chromosome 
10. The flanking sequences were used to isolate ho- 
mologous clones from the wild-type genome. The 
wild-type clones did not overlap, implying that a 
deletion had accompanied integration of the trans- 
genic DNA. To determine the size of the deletion, 
a restriction fragment analysis was done, using 
pulsed-field gel electrophoresis. The results indi- 
cate that there was a deletion of —75 kb and that 
the deleted region contains a G-C-rich region, 
which could potentially represent the 5' end of the 
downless gene. Bidirectional chromosomal walking 
from both flanks is in progress, and —60 kb of ge- 
nomic sequences has been isolated. The genomic 
sequences are being screened for evolutionary con- 
servation, and the conserved sequences are being 
used as probes to screen a newborn skin cDNA li- 
brary. 
11. spermless. 
In another transgenic family, integration of the 
transgenic DNA has inactivated a gene essential for 
normal sperm development. The mutation is reces- 
sive and leads to sterility in homozygous transgenic 
males. The fertility of the homozygous females is 
not affected. The mutation appears to block sper- 
matogenesis at a specific stage of spermatid devel- 
opment. Mitotic and meiotic divisions appear to be 
unaffected, but development ceases at the round 
spermatid stage, prior to the onset of sperm tail syn- 
thesis. The localized and stage-specific nature of the 
defect suggests that a gene essential for a unique step 
in sperm maturation has been inactivated. 
The genomic sequences flanking one side of the 
transgenic insert have now been cloned. These se- 
quences were used for a restriction fragment length 
polymorphism (RFLP) analysis, which confirmed 
that the sterile males were homozygous for the 
transgenic insert. The sequences have also been 
mapped adjacent to the retinoblastoma locus on 
mouse chromosome 14. Current experiments are 
directed toward the cloning of the genomic se- 
quences flanking the other end of the transgenic in- 
sert and the cloning of the corresponding regions 
of the wild-type genome. 
III. Truncated SV40 Early Region. 
Transgenic mice that express the full-length SV40 
T antigen in the lens develop lens tumors. An inter- 
esting transgenic family was found to have inte- 
grated a truncated version of the T antigen. The 
mice show lens ablation rather than lens tumor for- 
mation. The truncation appears to have converted 
the T antigen from an inducer of cell proliferation 
to an inhibitor of cell proliferation. The transgenic 
insert, along with the adjacent mouse genomic se- 
quences, has been cloned and sequenced. The 
T antigen-coding region was found to be truncated 
near amino acid 200 and fused to an open reading 
frame of 21 amino acids in the genomic sequences. 
In collaboration with Dr. Janet Butel, immuno- 
precipitation experiments revealed the presence of 
a protein of the appropriate size in lens extracts 
from the transgenic mice. The truncated T antigen 
retains the region that interacts with the retinoblas- 
toma (RB) protein, while the region that binds to 
p53 has been lost. This observation suggests possi- 
ble models for the mechanism of action of the al- 
tered T antigen. For instance, the modified T anti- 
gen may stabilize the antiproliferative function of 
the RB protein (perhaps by blocking phosphoryla- 
tion). As a consequence, mitosis might be inhibited, 
resulting in lens ablation. Experiments have re- 
cently been initiated using tissue culture cells to 
test this model. 
Continued 
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