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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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Schematic representation of three αA-crystallin transgene constructs. (A) A schematic diagram of the mouse Cryaa locus (chromosome 17) and its adjacent loci, U2af1 and Snf1lk. The 148 kb BAC Clone RP-23-465G4 is shown in red. The XmaI-SpeI sites delineate 15 kb of the Cryaa locus. Exons in the Cryaa gene (black box), DCR1 and DCR3 (orange box), centromere, cen; telomere, tel; Exon, ex; rodent specific Cryaa exon, Ins. (B) Modification of αA-BAC using the λ prophage system for homologous recombination [25]. (C) Generation of αA-BAC(ΔDCR3) using a shuttle vector [26]. (D) Diagrammatic representation of the 15 kb portion of the mouse αA-crystallin locus with an EGFP insert (see panel (C)) marked by unique XmaI and SpeI restriction sites.
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Figure 2: Schematic representation of three αA-crystallin transgene constructs. (A) A schematic diagram of the mouse Cryaa locus (chromosome 17) and its adjacent loci, U2af1 and Snf1lk. The 148 kb BAC Clone RP-23-465G4 is shown in red. The XmaI-SpeI sites delineate 15 kb of the Cryaa locus. Exons in the Cryaa gene (black box), DCR1 and DCR3 (orange box), centromere, cen; telomere, tel; Exon, ex; rodent specific Cryaa exon, Ins. (B) Modification of αA-BAC using the λ prophage system for homologous recombination [25]. (C) Generation of αA-BAC(ΔDCR3) using a shuttle vector [26]. (D) Diagrammatic representation of the 15 kb portion of the mouse αA-crystallin locus with an EGFP insert (see panel (C)) marked by unique XmaI and SpeI restriction sites.

Mentions: Prior studies identified two distal control regions, DCR1 and DCR3 (Fig. 2A), that, in combination with a 1.9 kb promoter fragment, recapitulate most aspects of transcriptional regulation of αA-crystallin [23]. However, in these studies, the onset of EGFP expression was between E11-E11.5, which is at least 12 hours after the onset of endogenous αA-crystallin expression (see Fig. 1B). This finding suggests other regulatory regions could be required. To address this, we generated a BAC transgenic by modifying a 148 kb BAC encompassing the αA-crystallin gene (see Fig. 2A) through an in frame insertion of EGFP into the third exon, by homologous recombination according to [25] (see Fig. 2B). EGFP was inserted after V146, close to the C-end of the 173 amino acid αA-crystallin. Four founders were generated, but only three were studied due to breeding difficulties encountered with the fourth founder. All founders had visually green eyes, and transgene integrations were subsequently confirmed by genotyping. The copy number of the transgenic lines was determined by qPCR (Table 1) as described in Methods.


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)

Schematic representation of three αA-crystallin transgene constructs. (A) A schematic diagram of the mouse Cryaa locus (chromosome 17) and its adjacent loci, U2af1 and Snf1lk. The 148 kb BAC Clone RP-23-465G4 is shown in red. The XmaI-SpeI sites delineate 15 kb of the Cryaa locus. Exons in the Cryaa gene (black box), DCR1 and DCR3 (orange box), centromere, cen; telomere, tel; Exon, ex; rodent specific Cryaa exon, Ins. (B) Modification of αA-BAC using the λ prophage system for homologous recombination [25]. (C) Generation of αA-BAC(ΔDCR3) using a shuttle vector [26]. (D) Diagrammatic representation of the 15 kb portion of the mouse αA-crystallin locus with an EGFP insert (see panel (C)) marked by unique XmaI and SpeI restriction sites.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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Figure 2: Schematic representation of three αA-crystallin transgene constructs. (A) A schematic diagram of the mouse Cryaa locus (chromosome 17) and its adjacent loci, U2af1 and Snf1lk. The 148 kb BAC Clone RP-23-465G4 is shown in red. The XmaI-SpeI sites delineate 15 kb of the Cryaa locus. Exons in the Cryaa gene (black box), DCR1 and DCR3 (orange box), centromere, cen; telomere, tel; Exon, ex; rodent specific Cryaa exon, Ins. (B) Modification of αA-BAC using the λ prophage system for homologous recombination [25]. (C) Generation of αA-BAC(ΔDCR3) using a shuttle vector [26]. (D) Diagrammatic representation of the 15 kb portion of the mouse αA-crystallin locus with an EGFP insert (see panel (C)) marked by unique XmaI and SpeI restriction sites.
Mentions: Prior studies identified two distal control regions, DCR1 and DCR3 (Fig. 2A), that, in combination with a 1.9 kb promoter fragment, recapitulate most aspects of transcriptional regulation of αA-crystallin [23]. However, in these studies, the onset of EGFP expression was between E11-E11.5, which is at least 12 hours after the onset of endogenous αA-crystallin expression (see Fig. 1B). This finding suggests other regulatory regions could be required. To address this, we generated a BAC transgenic by modifying a 148 kb BAC encompassing the αA-crystallin gene (see Fig. 2A) through an in frame insertion of EGFP into the third exon, by homologous recombination according to [25] (see Fig. 2B). EGFP was inserted after V146, close to the C-end of the 173 amino acid αA-crystallin. Four founders were generated, but only three were studied due to breeding difficulties encountered with the fourth founder. All founders had visually green eyes, and transgene integrations were subsequently confirmed by genotyping. The copy number of the transgenic lines was determined by qPCR (Table 1) as described in Methods.

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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