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Transcriptional regulation of mouse alpha A-crystallin gene in a 148kb Cryaa BAC and its derivates.

Wolf L, Yang Y, Wawrousek E, Cvekl A - BMC Dev. Biol. (2008)

Bottom Line: The number of cells expressing alphaA-crystallin in the lens pit was higher compared to the number of cells expressing EGFP.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Departments of Ophthalmology and Visual Sciences, Bronx, NY 10461, USA. lwolf@aecom.yu.edu

ABSTRACT

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.

Conclusion: 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.

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αA-BAC(ΔDCR3) transgene expression in the lens. Expression of EGFP from αA-BAC(ΔDCR3) is first observed at E10.5 in the lens pit (A, F). EGFP is expressed in the lens vesicle at E11.5 (Fig. B, G) and within the differentiating primary fiber cells at E12.5 (C, H), E14.5 (D, I) and in PND 1 lens (E, J). Fluorescent nuclear DAPI staining is blue and cytoskeletal staining is red. Lens fiber cells, f; lens pit, lp; lens vesicle, lv. Scale bar = 100 μm.
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Figure 4: αA-BAC(ΔDCR3) transgene expression in the lens. Expression of EGFP from αA-BAC(ΔDCR3) is first observed at E10.5 in the lens pit (A, F). EGFP is expressed in the lens vesicle at E11.5 (Fig. B, G) and within the differentiating primary fiber cells at E12.5 (C, H), E14.5 (D, I) and in PND 1 lens (E, J). Fluorescent nuclear DAPI staining is blue and cytoskeletal staining is red. Lens fiber cells, f; lens pit, lp; lens vesicle, lv. Scale bar = 100 μm.

Mentions: Similar to αA-BAC, EGFP expression is first evident in the lens pit at E10.5 (Fig. 4A, F) in one of the three αA-BAC (ΔDCR3) lines. Expression of EGFP is also detected in the lens vesicle (Fig. 4B, G) and is upregulated considerably in the differentiating lens fiber cells at E12.5 (Fig. 4C, H). As development progresses, EGFP is intensely expressed in the lens fiber cells at E14.5 (Fig. 4D, I; Additional file 1C) and PND 1 (Fig. 4E, J; Additional file 1D). Lower EGFP is also evident in some lens epithelial cells at these stages (Additional file 1C, D). The remaining two lines (see Table 1 and 2) have a delayed onset of EGFP expression, first found in the lens vesicle stage of development (data not shown).


Transcriptional regulation of mouse alpha A-crystallin gene in a 148kb Cryaa BAC and its derivates.

Wolf L, Yang Y, Wawrousek E, Cvekl A - BMC Dev. Biol. (2008)

αA-BAC(ΔDCR3) transgene expression in the lens. Expression of EGFP from αA-BAC(ΔDCR3) is first observed at E10.5 in the lens pit (A, F). EGFP is expressed in the lens vesicle at E11.5 (Fig. B, G) and within the differentiating primary fiber cells at E12.5 (C, H), E14.5 (D, I) and in PND 1 lens (E, J). Fluorescent nuclear DAPI staining is blue and cytoskeletal staining is red. Lens fiber cells, f; lens pit, lp; lens vesicle, lv. Scale bar = 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2567317&req=5

Figure 4: αA-BAC(ΔDCR3) transgene expression in the lens. Expression of EGFP from αA-BAC(ΔDCR3) is first observed at E10.5 in the lens pit (A, F). EGFP is expressed in the lens vesicle at E11.5 (Fig. B, G) and within the differentiating primary fiber cells at E12.5 (C, H), E14.5 (D, I) and in PND 1 lens (E, J). Fluorescent nuclear DAPI staining is blue and cytoskeletal staining is red. Lens fiber cells, f; lens pit, lp; lens vesicle, lv. Scale bar = 100 μm.
Mentions: Similar to αA-BAC, EGFP expression is first evident in the lens pit at E10.5 (Fig. 4A, F) in one of the three αA-BAC (ΔDCR3) lines. Expression of EGFP is also detected in the lens vesicle (Fig. 4B, G) and is upregulated considerably in the differentiating lens fiber cells at E12.5 (Fig. 4C, H). As development progresses, EGFP is intensely expressed in the lens fiber cells at E14.5 (Fig. 4D, I; Additional file 1C) and PND 1 (Fig. 4E, J; Additional file 1D). Lower EGFP is also evident in some lens epithelial cells at these stages (Additional file 1C, D). The remaining two lines (see Table 1 and 2) have a delayed onset of EGFP expression, first found in the lens vesicle stage of development (data not shown).

Bottom Line: The number of cells expressing alphaA-crystallin in the lens pit was higher compared to the number of cells expressing EGFP.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Departments of Ophthalmology and Visual Sciences, Bronx, NY 10461, USA. lwolf@aecom.yu.edu

ABSTRACT

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.

Conclusion: 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.

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Related in: MedlinePlus