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Blood, Vol. 113, Issue 6, 1340-1349, February 5, 2009
Mir-144 selectively regulates embryonic  -hemoglobin synthesis during primitive erythropoiesis
Blood Fu et al.
113: 1340
Supplemental materials for: Fu et al
Files in this Data Supplement:
- Figure S7. Quantitative analysis of gene expression in miR-144 knockdown embryos (JPG, 48 KB)
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Quantitative real-time RT-PCR analysis of genes in the embryos injected with either miR-144 or control LNA. Data were presented as means ± SEM from three independent experiments. Zebrafish gapdh housekeeping gene was used as an internal control.
- Figure S8. Endogenous miR-144 inhibits EGFP reporter activity by targeting klfd 3′UTR (JPG, 133 KB)
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(A,B) EGFP mRNAs without (A) or with (B) two perfectly complementary miR-144 targeting sites (2×PT) were co-injected with either miR-144 control duplex or miR-144 duplex into one-cell stage embryos. The dsRed mRNAs was co-injected as a control (bottom panels). Sequences of miR-144 duplex and PT were shown below. (C–F) EGFP mRNAs with two or three miR-144 recognition element (2× or 3×MRE) from 3′UTR of lmo2 (C), alas2 (D), klf12 (E) and klfd (F), were co-injected with either miR-144 control duplex or miR-144 duplex into one-cell stage embryos. The dsRed mRNAs was co-injected as a control (bottom panels). The MRE sequences complementary to the miR-144 were shown below.
- Figure S9. miR-144 targets klfd in other Tg(zgata-1:EGFP-klfd 3′UTR) transgenic lines (JPG, 54.2 KB)
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EGFP mRNAs and protein levels were decreased in the ICM of F1 24 hpf embryos derived from line 1, line 6 and line 8 of the Tg(zgata-1:EGFP-klfd 3′UTR).
- Figure S10. Effects of klfd overexpression on hematopoietic gene expression by WISH (JPG, 64.3 KB)
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Forced expression of klfd mRNAs slightly increases, if any, alas2 expression without affecting the expression of gata-1, mpo, myo D, and krox 20 genes.
- Figure S11. Knockdown efficiency of klfd morpholino oligonucleotides (JPG, 58.6 KB)
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(A) Schematic presentation of the reporter plasmid used to test the efficiency of morpholino knockdown. The full-length 5′UTR and immediate 21-nt downstream of translational start codon (ATG) of klfd was fused to the ATG-deleted open reading frame of EGFP in the pCS2+ plasmid. The bar denotes the targeted site recognized by morpholino. (B) Co-injection of EGFP reporter mRNAs with either 5-mismatch control or klfd morpholino oligonucleotides into wild-type embryos. Fluorescent steromicroscope shows EGFP expression at 22 hpf. Co-injection of DsRed mRNAs was used a control below.
- Figure S12. Syntenic analysis of zebrafish klfd gene (JPG, 58.7 KB)
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The klfd gene resides within a region of zebrafish chromosome 5 that is syntenic with the human klf1 locus. (Left) Nine genes including KLF1 and KLF2 are located within a genomic region on human chromosome 19p, according to the latest version of the human genome draft (http://genome.ucsc.edu). (Right) Six (including klfd) and three (including klf2a) zebrafish homologues of these genes are listed according to their map positions on chromosomes 6 and 22, respectively (http://www.ensembl.org).
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