A BAC-bacterial recombination method to generate physically linked multiple gene reporter DNA constructs.
BACKGROUND
Reporter gene mice are valuable animal models for biological research providing a gene expression readout that can contribute to cellular characterization within the context of a developmental process. With the advancement of bacterial recombination techniques to engineer reporter gene constructs from BAC genomic clones and the generation of optically distinguishable fluorescent protein reporter genes, there is an unprecedented capability to engineer more informative transgenic reporter mouse models relative to what has been traditionally available.


RESULTS
We demonstrate here our first effort on the development of a three stage bacterial recombination strategy to physically link multiple genes together with their respective fluorescent protein (FP) reporters in one DNA fragment. This strategy uses bacterial recombination techniques to: (1) subclone genes of interest into BAC linking vectors, (2) insert desired reporter genes into respective genes and (3) link different gene-reporters together. As proof of concept, we have generated a single DNA fragment containing the genes Trap, Dmp1, and Ibsp driving the expression of ECFP, mCherry, and Topaz FP reporter genes, respectively. Using this DNA construct, we have successfully generated transgenic reporter mice that retain two to three gene readouts.


CONCLUSIONS
The three stage methodology to link multiple genes with their respective fluorescent protein reporter works with reasonable efficiency. Moreover, gene linkage allows for their common chromosomal integration into a single locus. However, the testing of this multi-reporter DNA construct by transgenesis does suggest that the linkage of two different genes together, despite their large size, can still create a positional effect. We believe that gene choice, genomic DNA fragment size and the presence of endogenous insulator elements are critical variables.
Maye
Peter
P
Department of Reconstructive Sciences, Center for Regenerative Medicine, University of Connecticut Health Center, Farmington, CT, USA. pmaye@neuron.uchc.edu
Stover
Mary Louise
ML
Liu
Yaling
Y
Rowe
David W
DW
Gong
Shiaochin
S
Lichtler
Alexander C
AC
D000818:Animals; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D018628:Gene Dosage; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors; D051379:Mice; D008822:Mice, Transgenic; D016254:Mutagenesis, Insertional / Q000379:methods*; D011995:Recombination, Genetic
D004247:DNA