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Novel Genes Critical for Hypoxic Preconditioning in Zebrafish Are Regulators of Insulin and Glucose Metabolism.

Manchenkov T, Pasillas MP, Haddad GG, Imam FB - G3 (Bethesda) (2015)

Bottom Line: Severe hypoxia is a common cause of major brain, heart, and kidney injury in adults, children, and newborns.However, mild hypoxia can be protective against later, more severe hypoxia exposure via "hypoxic preconditioning," a phenomenon that is not yet fully understood.Using a functional genomic approach, we used this zebrafish model to identify and validate five novel hypoxia-protective genes, including irs2, crtc3, and camk2g2, which have been previously implicated in metabolic regulation.

View Article: PubMed Central - PubMed

Affiliation: Division of Neonatology, University of California San Diego School of Medicine, La Jolla, California 92093.

No MeSH data available.


Related in: MedlinePlus

irs2 is required for acute hypoxic stress buffering. (A) h-MO knockdown survival is shown in comparison to ctl-MO survival under the following hypoxia exposures: none (MO injection alone); brief sH(1d:5h); and moderate sH(1.5d:30h). irs2 inhibition significantly impairs moderate sH survival (P = 0.0029). (B) qRT-PCR validation of targeted splice h-MO knockdown is shown for ttll11, camk2g2, btr01, ncam2, and crct3. Note that only a translation-blocking MO was obtainable for irs2 because it only contains a single intron that is unsuitable for MO targeting; therefore, qPCR was not performed. n = 2 to 8 biological replicates with SEM for (A) and (B).
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fig4: irs2 is required for acute hypoxic stress buffering. (A) h-MO knockdown survival is shown in comparison to ctl-MO survival under the following hypoxia exposures: none (MO injection alone); brief sH(1d:5h); and moderate sH(1.5d:30h). irs2 inhibition significantly impairs moderate sH survival (P = 0.0029). (B) qRT-PCR validation of targeted splice h-MO knockdown is shown for ttll11, camk2g2, btr01, ncam2, and crct3. Note that only a translation-blocking MO was obtainable for irs2 because it only contains a single intron that is unsuitable for MO targeting; therefore, qPCR was not performed. n = 2 to 8 biological replicates with SEM for (A) and (B).

Mentions: Accordingly, antisense MO oligonucleotides were designed and tested against 10 representative hypoxia-responsive genes among those with the highest differential expression shared between gastrulation and segmentation embryos and those with highest egln3 correlation (Figure 3, Figure 4, Table 1). Morphant embryos for these candidate hypoxia-protective genes were exposed to both brief and moderate hypoxia protocols, sH(1d:5h) and sH(1.5d:30h), because these protocols caused low phenotypic injury in controls and were therefore poised to detect exacerbations of lethality due to MO inhibition of hypoxia-protective gene function (<10% and <20%, respectively; Figure 3B, Figure 4A). At least 100 embryos were injected per MO for each gene analyzed. Knockdown of the intended MO target for splice-blocking MOs was validated by qPCR, because the predicted aberrant splice product with a retained intron is often too large to amplify or more rapidly degraded due to nonsense-mediated decay (Bill et al. 2009; Draper et al. 2001; Knapp et al. 2006). Accordingly, targeted MO knockdown was found to decrease gene expression levels down to ∼15–30% of normal (Figure 4B).


Novel Genes Critical for Hypoxic Preconditioning in Zebrafish Are Regulators of Insulin and Glucose Metabolism.

Manchenkov T, Pasillas MP, Haddad GG, Imam FB - G3 (Bethesda) (2015)

irs2 is required for acute hypoxic stress buffering. (A) h-MO knockdown survival is shown in comparison to ctl-MO survival under the following hypoxia exposures: none (MO injection alone); brief sH(1d:5h); and moderate sH(1.5d:30h). irs2 inhibition significantly impairs moderate sH survival (P = 0.0029). (B) qRT-PCR validation of targeted splice h-MO knockdown is shown for ttll11, camk2g2, btr01, ncam2, and crct3. Note that only a translation-blocking MO was obtainable for irs2 because it only contains a single intron that is unsuitable for MO targeting; therefore, qPCR was not performed. n = 2 to 8 biological replicates with SEM for (A) and (B).
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Related In: Results  -  Collection

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fig4: irs2 is required for acute hypoxic stress buffering. (A) h-MO knockdown survival is shown in comparison to ctl-MO survival under the following hypoxia exposures: none (MO injection alone); brief sH(1d:5h); and moderate sH(1.5d:30h). irs2 inhibition significantly impairs moderate sH survival (P = 0.0029). (B) qRT-PCR validation of targeted splice h-MO knockdown is shown for ttll11, camk2g2, btr01, ncam2, and crct3. Note that only a translation-blocking MO was obtainable for irs2 because it only contains a single intron that is unsuitable for MO targeting; therefore, qPCR was not performed. n = 2 to 8 biological replicates with SEM for (A) and (B).
Mentions: Accordingly, antisense MO oligonucleotides were designed and tested against 10 representative hypoxia-responsive genes among those with the highest differential expression shared between gastrulation and segmentation embryos and those with highest egln3 correlation (Figure 3, Figure 4, Table 1). Morphant embryos for these candidate hypoxia-protective genes were exposed to both brief and moderate hypoxia protocols, sH(1d:5h) and sH(1.5d:30h), because these protocols caused low phenotypic injury in controls and were therefore poised to detect exacerbations of lethality due to MO inhibition of hypoxia-protective gene function (<10% and <20%, respectively; Figure 3B, Figure 4A). At least 100 embryos were injected per MO for each gene analyzed. Knockdown of the intended MO target for splice-blocking MOs was validated by qPCR, because the predicted aberrant splice product with a retained intron is often too large to amplify or more rapidly degraded due to nonsense-mediated decay (Bill et al. 2009; Draper et al. 2001; Knapp et al. 2006). Accordingly, targeted MO knockdown was found to decrease gene expression levels down to ∼15–30% of normal (Figure 4B).

Bottom Line: Severe hypoxia is a common cause of major brain, heart, and kidney injury in adults, children, and newborns.However, mild hypoxia can be protective against later, more severe hypoxia exposure via "hypoxic preconditioning," a phenomenon that is not yet fully understood.Using a functional genomic approach, we used this zebrafish model to identify and validate five novel hypoxia-protective genes, including irs2, crtc3, and camk2g2, which have been previously implicated in metabolic regulation.

View Article: PubMed Central - PubMed

Affiliation: Division of Neonatology, University of California San Diego School of Medicine, La Jolla, California 92093.

No MeSH data available.


Related in: MedlinePlus