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The main reason for abnormal development of cloned animals or embryos, and inefficient animal cloning, is a poor understanding of the reprogramming mechanism. To better comprehend reprogramming and subsequent generation of pluripotent stem cells, we must investigate factors related to reprogramming of somatic cells as nuclear donors. As we know, fam60al (family with sequence similarity 60, member A, like) is a coding gene only found in zebrafish and frog (Xenopus laevis) among vertebrates. However, until now, its functions have remained unknown. Here, we generated a zebrafish fam60al-/- mutant line using transcription activator-like effector nucleases (TALENs), and found that both nanog and klf4b expression significantly decreased while myca expression significantly increased in fam60al-/- mutant embryos. Concurrently, we also uncovered that in developmentally arrested embryos of somatic cell nuclear transfer, nanog, klf4b and myca expression was down-regulated, accompanying a decrease of fam60al expression. Interestingly, we identified a long noncoding RNA (lncRNA) of fam60al, named fam60al-AS, which negatively regulated fam60al by forming double-stranded RNA (dsRNA). RNase protection assay and real-time PCR confirmed these findings. Taken together, these results suggest that fam60al is a novel factor related to the reprogramming of somatic cell nuclear transfer in zebrafish, which is regulated by its reverse lncRNA.
Figure 1. Fam60al and its antisense transcript, fam60al-AS, in zebrafish. (A) Fam60al and fam60al-AS gene structure. Red marks the antisense overlapping sequence. CPC predicted fam60al-AS as an lncRNA. (B) Dot blot of the shield stage, 24 hpf and 72 hpf embryos, using sense and antisense probes of fam60al and fam60al-AS. Antisense probes have hybridization signal, while sense probes have no hybridization signal. (C) WISH of 24hpf and 72hpf embryos using sense and antisense probes of fam60al and fam60al-AS. Antisense probes have hybridization signal, sense probes have no hybridization signal. (scale bar: 200 μm).
Figure 2. Fam60al and fam60al-AS expression at different developmental stages in zebrafish. A: RT-PCR analysis of fam60al and fam60al-AS expression during early development, normalized to β-actin. B: RT-qPCR analysis of fam60al and fam60al-AS expression. The 2-ÎÎct method was used for measuring the expression levels and normalized to β-actin. 256-cell stage fam60al-AS expression level was set to 1.
Figure 3. Fam60al-AS negatively regulates fam60al expression by forming dsRNA. A: RT-qPCR analysis of fam60al in fam60al-AS overexpressed embryos. Overexpression of fam60al-AS at the sphere stage, resulted in down-regulation of fam60al expression. *P < 0.05; ** P < 0.01; WT: wild type; OE: over expression. B: RNase protection assay analyzing the dsRNA formed in the overlapping region of fam60al and fam60al-AS at the sphere stage and 24 hpf. PCR bands were detected at the sphere stage and 24hpf with RNase A or RNaseOutTM digestion, while no PCR band was observed at the sphere stage and 24hpf following denaturing at 95°C and digestion with RNase A.
Figure 4. Establish of fam60al knockout line with TALENs in zebrafish. A: Schematic showing fam60al gene knockout target sites. Target 1 was on exon 2 and Target 2 on exon 4. B: Primer design sites, transcript map and protein coding sequence for fam60al in fam60al-/- line. Primers F1 and R1 were designed to amplify the fam60al gene fragment deleted 2091bp in fam60alw/- and fam60al-/-. Primers F1 and R2 were designed to amplify the wild type fam60al gene fragment in fam60alw/w and fam60alw/-. Fam60al-/- encoded a premature termination 33 amino acids peptide. C: PCR results of different genetype zebrafish. Primer set 1: amplified fragment of primers F1 and R1, Primer set 2: amplified fragment of primers F1 and R2, M: DNA marker, *: fam60al-/-, #: fam60alw/w, and the rest for fam60alw/-.
Figure 5. mRNA expression levels of fam60al, fam60al-AS, nanog, klf4b and myca in wild type embryos and fam60al mutant embryos at the sphere and shield stage. The 2-Îct method was used for measuring the expression levels. β-actin was used as the internal control. *P < 0.05, **P < 0.01.
Figure 6. Developmental profiles of nuclear transplants at different stages. A: The average number of different batches of kidney cell nuclear transfer embryos. N(failed) = 77.34 ± 5.65, N(sphere) = 8.03 ± 1.83, N(shield) = 0.52 ± 0.54. B: Percentage of kidney cell nuclear transfer embryos at different stages. SCNT embryos failed to cleave. SCNT embryos arrested at the sphere stage. SCNT embryos successfully developed at the shield stage.
Figure 7. RT-qPCR detected the expression in SCNT embryos and wild type embryos at the sphere and shield stage. The 2-Îct method was used for measuring the expression levels and β-actin was used as the internal control. *P < 0.05, **P < 0.01. WT: wild type; SCNT: somatic cell nuclear transfer.
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