Transcriptional regulation of mouse alpha A-crystallin gene in a 148kb Cryaa BAC and its derivates.
BACKGROUND
alphaA-crystallin is highly expressed in the embryonic, neonatal and adult mouse lens. Previously, we identified two novel distal control regions, DCR1 and DCR3. DCR1 was required for transgenic expression of enhanced green fluorescent protein, EGFP, in lens epithelium, whereas DCR3 was active during "late" stages of lens primary fiber cell differentiation. However, the onset of transgenic EGFP expression was delayed by 12-24 hours, compared to the expression of the endogenous Cryaa gene.


RESULTS
Here, we used bacterial artificial chromosome (BAC) and standard transgenic approaches to examine temporal and spatial regulation of the mouse Cryaa gene. Two BAC transgenes, with EGFP insertions into the third coding exon of Cryaa gene, were created: the intact alphaA-crystallin 148 kb BAC (alphaA-BAC) and alphaA-BAC(DeltaDCR3), which lacks approximately 1.0 kb of genomic DNA including DCR3. Expression of EGFP in the majority of both BAC transgenics nearly recapitulated the endogenous expression pattern of the Cryaa gene in lens, but not outside of the lens. The number of cells expressing alphaA-crystallin in the lens pit was higher compared to the number of cells expressing EGFP. Next, we generated additional lines using a 15 kb fragment of alphaA-crystallin locus derived from alphaA-BAC(DeltaDCR3), 15 kb Cryaa/EGFP. A 15 kb region of Cryaa/EGFP supported the expression pattern of EGFP also in the lens pit. However, co-localization studies of alphaA-crystallin and EGFP indicated that the number of cells that showed transgenic expression was higher compared to cells expressing alphaA-crystallin in the lens pit.


CONCLUSIONS
We conclude that a 148 kb alphaA-BAC likely contains all of the regulatory regions required for alphaA-crystallin expression in the lens, but not in retina, spleen and thymus. In addition, while the 15 kb Cryaa/EGFP region also supported the expression of EGFP in the lens pit, expression in regions such as the hindbrain, indicate that additional genomic regions may play modulatory functions in regulating extralenticular alphaA-crystallin expression. Finally, deletion of DCR3 in either alphaA-BAC(DeltaDCR3) or Cryaa (15 kb) transgenic mice result in EGFP expression patterns that are consistent with DCR's previously established role as a distal enhancer active in "late" primary lens fiber cells.
Wolf
Louise
L
The Departments of Ophthalmology and Visual Sciences, Bronx, NY 10461, USA. lwolf@aecom.yu.edu
Yang
Ying
Y
Wawrousek
Eric
E
Cvekl
Ales
A
D000818:Animals; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental / Q000502:physiology*; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007908:Lens, Crystalline / Q000196:embryology / Q000378:metabolism; D051379:Mice; D014158:Transcription, Genetic / Q000502:physiology*; D019076:Transgenes; D038202:alpha-Crystallin A Chain / Q000096:biosynthesis / Q000235:genetics* / Q000502:physiology
D038202:alpha-Crystallin A Chain; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins