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Am J Physiol Heart Circ Physiol (February 23, 2007). doi:10.1152/ajpheart.00060.2007
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Submitted on January 15, 2007
Accepted on February 20, 2007

Efficient transgenic rat production by a lentiviral vector

Mieczyslaw Michalkiewicz1*, Teresa Michalkiewicz1, Aron M Geurts2, Richard J. Roman3, Glenn R Slocum1, Oded Singer4, Dorothee Weihrauch5, Andrew S Greene6, Mary L Kaldunski7, Inder M Verma4, Howard J Jacob8, and Allen W. Cowley Jr.7

1 Department of Physiology, Medical College of Wisconsin, Milwaukee, United States
2 Human and Molecular Genetics Center, United States; Department of Physiology, Medical College of Wisconsin, Milwaukee, United States
3 Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin, United States; Kidney Disease Center, United States
4 Laboratory of Genetics, The Salk Institute, La Jolla, California, United States
5 Dept of Anesthesiology, Medical College of Wisconsin, Milwaukee, Wisconsin, United States
6 Biotechnology and Bioengineering Center, Medical College of Wisconsin, Milwaukee, Wisconsin, United States
7 Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin, United States
8 Human Molecular and Genetics Center, Medical College of Wisconsin, Milwaukee, Wisconsin, United States

* To whom correspondence should be addressed. E-mail: mmichalk{at}mcw.edu.

Transgenic lines of rats were produced by using a lentiviral construct for an enhanced green fluorescent protein (eGFP) driven by a chicken{beta}-actin promoter, cytomeglavirus enhancer and intronic sequences from rabbit {beta}-globin (CAG) to evaluate the usefulness of this approach for gene function studies. Fertilized eggs were collected from inbred Dahl S and outbred Sprague Dawley rats and approximately 100 pl of concentrated virus was microinjected into the perivitrelline space of one-cell embryos. Out of 121 embryos injected, 60 pups (49.6%) were born. Transgenic rates averaged 22% in Dahl S and 14% in SD rats. Copy number ranged from 1-4 in the founders and the inheritance of the transgene in a subsequent F1 population was 48.2%. The small number of insertion sites enabled us to derive inbred transgenic lines with a single copy of the transgene within one generation. Sequencing of each transgene insertion site revealed that they inserted as single copies with an insertional preference for the introns of genes. The CAG promoter drove high levels of expression of eGFP in the brain, kidney, heart and vasculature making it very suitable for exploring the cardiovascular function of newly discovered genes. The pattern of expression of eGFP was similar across five different F1 transgenic lines indicating that the expression of the transgene was independent of its chromosomal position. Thus, lentiviral transgenesis provides a powerful tool for the production of transgenic inbred rats and it will enhance the usefulness of this species in gene discovery and target validation studies.




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