Developmental Genetics 
Philippe M. Soriano, Ph.D., D.Sc. — Assistant Investigator 
Dr. Soriano is also Assistant Professor at the Institute for Molecular Genetics and Department of Cell 
Biology, Baylor College of Medicine. He obtained his Ph.D. degree in biochemistry and his D.Sc. degree 
from the University of Paris. He did postdoctoral research in France, and then with Rudolf Jaenisch in 
Germany and at the Whitehead Institute for Biomedical Research of the Massachusetts Institute of 
Technology, before joining the faculty at Baylor. Dr. Soriano is a Pew Scholar in the biomedical sciences. 
THE major aim of our research is to extend the 
understanding of early development of the 
mouse. Our approach is to create mice that are 
mutant in genes critical for this process. Two 
techniques are being used: random mutagenesis, 
which should discover unknown genes, and tar- 
geted mutagenesis in previously characterized 
genes for which no mutation is known. 
The random mutagenesis project is designed to 
identify developmentally regulated and critical 
genes in the embryo. Identification of the mu- 
tated gene in many of the classical mouse mu- 
tations has been difficult because there is no 
convenient tag by which to clone the muta- 
tion. Insertion mutagenesis in transgenic mice, 
wherein a gene is disrupted by introducing a for- 
eign fragment of DNA into the germline, is attrac- 
tive, since the transgene can serve as a tag. How- 
ever, only 1 out of 20 transgenic strains displays 
an overt phenotype, so the approach is laborious 
and time consuming. We have circumvented this 
problem by preselecting for mutations in embry- 
onic stem (ES) cells, which are then introduced 
into embryos to colonize the germline. 
The selection procedure we have used is 
termed "promoter trapping." In this method, a 
reporter gene is placed downstream of a splice 
acceptor, and the construct is then introduced 
into ES cells. Expression of the reporter gene can 
only originate from a flanking cellular promoter. 
Therefore transgenic mice derived from ES cells 
selected for such events can be used both to trace 
the activity of the tagged gene, by expression of 
the reporter gene, and to mutate the gene. The 
reporter gene we have used encodes a fusion pro- 
tein with both (8-galactosidase (;8-gal) and neo- 
mycin phosphotransferase (neo) activity. This al- 
lows one to select directly for promoter trap 
events, since neo confers resistance to the drug 
G418, and to follow the activity of the trapped 
gene by incubating the embryos in a dye that 
turns cells blue if |8-gal is present. 
Thirty-one transgenic lines have now been gen- 
erated using a retroviral promoter trap vector and 
have been examined both for patterns of expres- 
sion and for phenotype. We are particularly inter- 
ested in strains that exhibit restricted patterns of 
expression at gastrulation, a critical stage of de- 
velopment in the early mouse embryo. Twelve 
strains carry a recessive lethal mutation due to the 
promoter trap insertion, and one induces steril- 
ity. This demonstrates that the method is very 
valuable for isolating developmental mutants. 
Further analysis of one of the embryonic lethal 
strains suggests a mutation in a transcription fac- 
tor. The other strains, which do not display a phe- 
notype associated with the promoter trap event, 
may reflect mutations in nonessential genes. This 
first aspect of our research is supported by the 
National Institutes of Health. 
It is also possible to make specific mutations by 
targeting genes believed to play critical roles in 
development, based on their previous character- 
ization. This approach relies on the ability to in- 
troduce into ES cells mutant gene constructs that 
will recombine homologously with the normal 
gene, at the correct chromosomal location, there- 
fore creating a mutation in the gene of interest. 
Our initial efforts focused on the gene encoding 
c-src, the first proto-oncogene identified. 
Src is a protein-tyrosine kinase, broadly ex- 
pressed but at particularly high levels in neurons 
and in platelets. The src gene was knocked out in 
ES cells, and the mutation was transmitted 
through the germline. Surprisingly, animals ho- 
mozygous for the mutation do not die at an early 
stage, but develop osteopetrosis, a disease char- 
acterized by an impaired function of the osteo- 
clasts, the cells that normally resorb bone. As a 
result of the mutation, the mutant mice fail to 
undergo normal tooth eruption and have to be 
maintained on a soft food diet. We have been able 
to show that the defect lies in the osteoclasts 
proper, rather than in accessory cells such as os- 
teoblasts that condition osteoclast activity. How- 
ever, no defect is found in cells or tissues where 
src is most highly expressed. 
To explain the lack of a more severe pheno- 
type, we have tested the hypothesis that another 
5rc-related kinase may be compensating for loss 
of src in the mutant mice. Mice mutant in the 
closely related c-yes and fyn genes have therefore 
been generated by homologous recombination in 
ES cells. Neither of these mutations results in an 
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