Insertion of a Pax6 consensus binding site into the alphaA-crystallin promoter acts as a lens epithelial cell enhancer in transgenic mice.
OBJECTIVE
Although the murine alphaA-crystallin promoter is the most commonly used promoter for achieving transgene expression in the developing lens, this promoter directs transgene expression efficiently only in lens fiber cells. The purpose of the present study was to generate promoters capable of directing transgene expression to the entire lens but not to the corneal epithelium.


METHODS
Transgenic mice were generated with fragments of the murine alphaA- and alphaB-crystallin promoters, as well as with an alphaA-crystallin promoter engineered with the insertion of a Pax6 consensus binding site driving either human growth hormone (hGH) or Cre recombinase genes. hGH expression was evaluated by in situ hybridization and immunohistochemistry. Cre expression was revealed by x-gal staining after crossing Cre transgenic mice with a Cre reporter strain.


RESULTS
Within the lens, the -214/+38 alphaB-crystallin promoter fragment directed transgene expression in the lens epithelium, but not in fiber cells. The native -282/+43 alphaA-crystallin promoter drove transgene expression in the lens fiber cells of several independent lines of transgenic mice, but none of these mice demonstrated significant transgene expression in the lens epithelium. In contrast, the insertion of a 32-bp sequence containing a Pax6 consensus binding site into the -282/+43 alphaA-crystallin promoter reproducibly led to transgene expression in the lens epithelium as well as the lens fiber cells.


CONCLUSIONS
The inclusion of a Pax6 consensus binding site within the -282/+43 alphaA-crystallin promoter enhances the ability of this promoter to drive transgene expression in the lens epithelium.
Zhao
Haotian
H
Center for Molecular and Human Genetics, Columbus Children's Research Institute, Columbus, Ohio 43205, USA.
Yang
Ying
Y
Rizo
Christian M
CM
Overbeek
Paul A
PA
Robinson
Michael L
ML
D000818:Animals; D001665:Binding Sites; D016384:Consensus Sequence; D004742:Enhancer Elements, Genetic; D004847:Epithelial Cells / Q000378:metabolism*; D005136:Eye Proteins; D018507:Gene Expression Regulation, Developmental / Q000502:physiology*; D005822:Genetic Vectors; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D019382:Human Growth Hormone / Q000378:metabolism; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D019426:Integrases / Q000378:metabolism; D007908:Lens, Crystalline / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D016254:Mutagenesis, Insertional; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins; D019076:Transgenes; D014764:Viral Proteins / Q000378:metabolism; D038202:alpha-Crystallin A Chain / Q000235:genetics* / Q000378:metabolism
D005136:Eye Proteins; D018398:Homeodomain Proteins; D000071841:PAX6 Transcription Factor; C000605730:PAX6 protein, human; D051761:Paired Box Transcription Factors; C000605732:Pax6 protein, mouse; D012097:Repressor Proteins; D014764:Viral Proteins; D038202:alpha-Crystallin A Chain; D019382:Human Growth Hormone; C045073:Cre recombinase; D019426:Integrases