The major goal of this proposal is to identify genes which play important roles in mammalian embryogenesis. Our approach will combine a number of experimental systems and techniques. Two projects will involve generating random insertional mutations in mice. In the first, we will take advantage of the availability of a large pool of transgenic mouse strains. We plan to use a rapid in situ hybridization procedure to maximize the efficiency of detecting recessive mutations. In the second, cultures of embryonic stem cells (ES cells) will be used to generate mice carrying multiple copies of a retroviral vector sequence. We will use a variety of molecular and genetic screening methods to identify virally-induced dominant, recessive and sex linked mutations. These strategies should result in the generation of a number of new insertional mutations affecting the developmental process. Additionally, we plan to develop novel strategies for generating and recovering clones of ES cells carrying mutations in specific target genes. The growth factor gene IGF-II will serve as a model system for these studies. We will test several DNA constructs designed to promote gene disruption by homologous recombination, and to facilitate selection of ES cells in which such events have occurred. As a complementary approach we also plan to use a DNA amplification technique (polymerase chain reaction) to screen populations of retrovirally infected ES cultures for rare clones which carry a provirus inserted in the endogenous IGF-II gene. In both approaches, characterized ES cell clones will be used to generate chimeric mice, thus introducing the mutations into the germ line. Hopefully these studies will lead to the development of techniques whereby the role of any specific gene in the developing embryo can be tested.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
7R01HD025335-05
Application #
3326467
Study Section
Special Emphasis Panel (SRC (04))
Project Start
1992-09-01
Project End
1993-12-31
Budget Start
1992-09-01
Budget End
1992-12-31
Support Year
5
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Harvard University
Department
Type
Schools of Arts and Sciences
DUNS #
071723621
City
Cambridge
State
MA
Country
United States
Zip Code
02138
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Jeannotte, L; Ruiz, J C; Robertson, E J (1991) Low level of Hox1.3 gene expression does not preclude the use of promoterless vectors to generate a targeted gene disruption. off. Mol Cell Biol 11:5578-85
Lee, J E; Pintar, J; Efstratiadis, A (1990) Pattern of the insulin-like growth factor II gene expression during early mouse embryogenesis. Development 110:151-9