Chick delta1-crystallin enhancer influences mouse alphaA-crystallin promoter activity in transgenic mice.
OBJECTIVE
Both the -366/+43 and the -282/+43 mouse alphaA-crystallin (or alphaA) promoters have been effective at driving transgene expression in lens fiber cells, but not in lens epithelium. Because the chick delta1-crystallin gene is expressed in lens epithelial cells, an enhancer was borrowed from this gene and linked to the alphaA promoter. This heterogenic enhancer/promoter construct was tested in transgenic mice to see whether it was active in both lens epithelium and fiber cells while retaining lens specificity.


METHODS
The third intron of the chick delta1-crystallin gene, which contains a lens enhancer element, was added to the 5' end of the mouse alphaA promoter. We refer to this chimeric regulatory element as the deltaenalphaA promoter. To test its activity, we inserted coding sequences for five different genes. Transgenic mice were generated by pronuclear microinjection. Transgene expression patterns were analyzed by either X-gal staining, in situ hybridization or immunohistochemical staining.


RESULTS
When deltaenalphaA-lacZ transgenic embryos were stained with X-gal at embryonic day (E)11.5, beta-galactosidase activity was detected only in the eye. Histologic sections of the stained embryos revealed that lacZ was expressed exclusively in the lens, in both epithelial and fiber cells. Transgenic mice were also generated using either the original alphaA- or the new deltaenalphaA promoter linked to an insulin cDNA. In situ hybridizations confirmed that the short alphaA promoter targeted prenatal insulin expression specifically to the lens fiber cells, whereas the deltaenalphaA promoter was active in both lens epithelial and fiber cells. Developmental studies of the deltaenalphaA-insulin mice showed that the deltaenalphaA promoter became active at the lens pit stage and remained active in all lens cells, even at postnatal ages. The deltaenalphaA promoter also successfully directed expression of SV40 T-antigen (TAg), human E2F2, and dominant negative Sprouty2 (dn-Spry2) genes to lens epithelial and fiber cells. The lens specificity of the deltaenalphaA promoter was maintained in minigenes with different types of introns and polyadenylation signals.


CONCLUSIONS
A new lens-specific regulatory element was generated-the deltaenalphaA promoter, which can drive high levels of transgene expression in both lens epithelium and fiber cells throughout development. This modified promoter can be used for future transgenic studies of signal transduction and cell cycle regulation in lens epithelial cells.
Reneker
Lixing W
LW
Department of Ophthalmology, University of Missouri, Columbia, 65212, USA. renekerl@health.missouri.edu
Chen
Qin
Q
Bloch
Amy
A
Xie
Leike
L
Schuster
Gaby
G
Overbeek
Paul A
PA
D000818:Animals; D002645:Chickens; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D004847:Epithelial Cells / Q000378:metabolism; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D005786:Gene Expression Regulation; D005809:Genes, Regulator; D017403:In Situ Hybridization; D007908:Lens, Crystalline / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D011401:Promoter Regions, Genetic / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism*; D019076:Transgenes; D038202:alpha-Crystallin A Chain / Q000235:genetics*; D038207:delta-Crystallins / Q000235:genetics*
D014157:Transcription Factors; D038202:alpha-Crystallin A Chain; D038207:delta-Crystallins