Eye Development, Pigmentation, and 
Insertional Mutagenesis 
Paul A. Overbeek, Ph.D. — Assistant Investigator 
Dr. Overbeek is also Assistant Professor in the Department of Cell Biology, Institute for Molecular Genet- 
ics, and Division of Neuroscience at Baylor College of Medicine. He received his B.A. degree in chemistry 
from Kalamazoo College, his Ph.D. degree in cellular and molecular biology from the University of Mich- 
igan, and an M.B.A. degree from the University of Chicago. His postdoctoral research was done in the 
laboratory of Heiner Westphal at the NIH. 
TRANSGENIC mice are used in my laboratory 
as a model system to study mammalian gene 
regulation and embryonic development. Such 
mice are generated by microinjection of purified 
fragments of DNA into mouse embryos at the one- 
cell stage. The embryos are then transferred into 
the reproductive tracts of suitably prepared fe- 
male mice and allowed to develop to term. In a 
typical experiment, 20-30 percent of the new- 
born mice are found to have integrated the in- 
jected DNA stably into their genome. The new 
DNA is termed transgenic DNA. 
Through recombinant DNA technology, the 
DNA for microinjection can be specifically de- 
signed and assembled from previously character- 
ized fragments of DNA. The microinjected DNA, 
once it has integrated, is duplicated along with 
the rest of the genome at each cell division and is 
therefore present in all cells of the transgenic 
mice. The mice are analyzed in detail to deter- 
mine whether the new DNA causes changes in the 
development or behavior of the mice. 
The new DNA can work through at least two 
different mechanisms. If the new DNA is ex- 
pressed — i.e., if it encodes a protein — the char- 
acteristics of the mice can be altered in a "domi- 
nant" fashion. Alternatively, the transgenic DNA 
may cause a "recessive" mutation as a result of its 
novel position in the genome. When the new 
DNA integrates, the normal organization of the 
adjacent endogenous genes may be disrupted, re- 
sulting in recessive inactivation of those genes. 
Transgenic mice are typically analyzed for both 
dominant and recessive changes in their pattern 
of development. 
Much of our research is directed at studies of 
eye development. The lens of the eye is known to 
express a set of proteins called crystallins. The 
crystallins are essential for normal transparency 
of the lens, and certain crystallins are expressed 
exclusively in the lens, implying that lens-spe- 
cific regulatory sequences must be associated 
with those genes. In collaboration with Joram 
Piatigorsky and Heiner Westphal at the National 
Institutes of Health, experiments were done to 
identify lens-specific regulatory sequences. Re- 
combinant DNA techniques were used to link a 
putative crystallin regulatory sequence to coding 
sequences from a bacterial gene. The recombi- 
nant DNA construct was used to generate trans- 
genic mice. When the mice were assayed for ex- 
pression of the bacterial gene, activity was 
detected only in the lens. This result demon- 
strated that a small stretch of DNA located near 
the transcription initiation site of a crystallin 
gene was sufficient to provide lens-specific regu- 
lation of gene expression. 
A subsequent experiment was done to generate 
transgenic mice that would express an oncogene 
in their lenses. An oncogene encodes a protein 
that is thought to be capable of predisposing cells 
to cancer formation. A construct was made that 
contained the crystallin regulatory sequences 
linked to an oncogene from a tumor virus, and 
transgenic mice were generated. The mice dis- 
played dramatic bilateral cataracts, and their 
lenses were found to contain rapidly growing tu- 
mor cells. When the embryonic development of 
the lenses was studied, it became apparent that 
tumor cell proliferation began shortly after the 
onset of expression of the oncogene. These ex- 
periments showed that expression of the onco- 
gene was sufficient to induce tumor formation. 
These transgenic mice provide a model system in 
which to study the mechanisms by which an on- 
cogene can alter cellular differentiation and 
proliferation. 
Over the past two years, regulatory sequences 
that give cell-specific gene expression in other 
regions of the eye have been characterized. In 
collaboration with Gregory Liou, a promoter that 
is active in the photoreceptor cells of the retina 
has been identified. The promoter comes from a 
gene that encodes a retinoid-binding protein. 
This protein plays a role in the recycling of reti- 
nal, a vitamin A derivative that is essential for nor- 
mal vision. The identification of a photoreceptor- 
specific promoter will allow future studies of 
retinal function and development. 
Studies have also been done to identify a pro- 
moter that is active in the pigmented regions of 
the eye. In both mice and humans, loss of pig- 
537 
