Faculty Collaboration Database

Efficient transgenic rat production by a lentiviral vector. Am J Physiol Heart Circ Physiol 2007 Jul;293(1):H881-94

Date

02/27/2007

Pubmed ID

17322424

DOI

10.1152/ajpheart.00060.2007

Scopus ID

2-s2.0-34547112080 (requires institutional sign-in at Scopus site)   36 Citations

Abstract

A lentiviral construct for an enhanced green fluorescent protein (eGFP) driven by a chicken beta-actin promoter, cytomegalovirus enhancer, and intronic sequences from rabbit beta-globin (CAG) was used to produce transgenic lines of rats for evaluation of the usefulness of this approach in gene function studies. Fertilized eggs were collected from inbred Dahl S and outbred Sprague-Dawley rats, and approximately 100 pl of concentrated virus were microinjected into the perivitrelline space of one-cell embryos. Of 121 embryos injected, 60 pups (49.6%) were born. Transgenic rates averaged 22% in Dahl S and 14% in Sprague-Dawley rats. Copy number ranged from one to four in the founders, and the inheritance of the transgene in a subsequent F(1) 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 a preference for the introns of genes. The CAG promoter drove high levels of eGFP expression in brain, kidney, heart, and vasculature, making it very suitable for exploring the cardiovascular function of newly discovered genes. The pattern of eGFP expression was similar across five different F(1) 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 will enhance the usefulness of this species in gene discovery and target validation studies.

Author List

Michalkiewicz M, Michalkiewicz T, Geurts AM, Roman RJ, Slocum GR, Singer O, Weihrauch D, Greene AS, Kaldunski M, Verma IM, Jacob HJ, Cowley AW Jr

Authors

Allen W. Cowley Jr PhD Professor in the Physiology department at Medical College of Wisconsin
Aron Geurts PhD Professor in the Physiology department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Animals
Animals, Genetically Modified
Genetic Vectors
Lentivirus
Rats
Recombinant Proteins
Transfection