Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Int J Biol Sci
2018 Jan 11;141:78-86. doi: 10.7150/ijbs.22426.
Show Gene links
Show Anatomy links
Fam60al as a novel factor involved in reprogramming of somatic cell nuclear transfer in zebrafish (Danio rerio).
Hu H, Tao B, Chen J, Zhu Z, Hu W.
???displayArticle.abstract???
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-ΔΔct 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-Δct 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-Δct 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.
Bao,
The p53-induced lincRNA-p21 derails somatic cell reprogramming by sustaining H3K9me3 and CpG methylation at pluripotency gene promoters.
2015, Pubmed
Bao,
The p53-induced lincRNA-p21 derails somatic cell reprogramming by sustaining H3K9me3 and CpG methylation at pluripotency gene promoters.
2015,
Pubmed Bass,
Double-stranded RNA as a template for gene silencing.
2000,
Pubmed Beaujean,
Effect of limited DNA methylation reprogramming in the normal sheep embryo on somatic cell nuclear transfer.
2004,
Pubmed Bubenshchikova,
Nuclear transplants from adult somatic cells generated by a novel method using diploidized eggs as recipients in medaka fish (Oryzias latipes).
2008,
Pubmed Buganim,
Mechanisms and models of somatic cell reprogramming.
2013,
Pubmed Chen,
Studies on the developmental potentiality of cultured cell nuclei of fish.
2010,
Pubmed Cibelli,
Cloned transgenic calves produced from nonquiescent fetal fibroblasts.
1998,
Pubmed Cibelli,
The health profile of cloned animals.
2002,
Pubmed Derrien,
The GENCODE v7 catalog of human long noncoding RNAs: analysis of their gene structure, evolution, and expression.
2012,
Pubmed Durruthy-Durruthy,
The primate-specific noncoding RNA HPAT5 regulates pluripotency during human preimplantation development and nuclear reprogramming.
2016,
Pubmed Flynn,
Long noncoding RNAs in cell-fate programming and reprogramming.
2014,
Pubmed Guenther,
Transcriptional control of embryonic and induced pluripotent stem cells.
2011,
Pubmed Hastings,
Post-transcriptional regulation of thyroid hormone receptor expression by cis-acting sequences and a naturally occurring antisense RNA.
2000,
Pubmed Howe,
The zebrafish reference genome sequence and its relationship to the human genome.
2013,
Pubmed Huang,
Long non-coding antisense RNA KRT7-AS is activated in gastric cancers and supports cancer cell progression by increasing KRT7 expression.
2016,
Pubmed Jia,
Genome-wide computational identification and manual annotation of human long noncoding RNA genes.
2010,
Pubmed Katayama,
Antisense transcription in the mammalian transcriptome.
2005,
Pubmed Kato,
Eight calves cloned from somatic cells of a single adult.
1998,
Pubmed Kimmel,
Stages of embryonic development of the zebrafish.
1995,
Pubmed
,
Xenbase Knee,
Regulation of gene expression by natural antisense RNA transcripts.
1997,
Pubmed Kong,
CPC: assess the protein-coding potential of transcripts using sequence features and support vector machine.
2007,
Pubmed Le,
Enhanced telomere rejuvenation in pluripotent cells reprogrammed via nuclear transfer relative to induced pluripotent stem cells.
2014,
Pubmed Lee,
Dogs cloned from adult somatic cells.
2005,
Pubmed Lee,
Cloned zebrafish by nuclear transfer from long-term-cultured cells.
2002,
Pubmed Lee,
Nanog, Pou5f1 and SoxB1 activate zygotic gene expression during the maternal-to-zygotic transition.
2013,
Pubmed Li,
Expression of alternatively spliced FGF-2 antisense RNA transcripts in the central nervous system: regulation of FGF-2 mRNA translation.
2000,
Pubmed Li,
Cloned ferrets produced by somatic cell nuclear transfer.
2006,
Pubmed Liu,
Inheritable and precise large genomic deletions of non-coding RNA genes in zebrafish using TALENs.
2013,
Pubmed Loewer,
Large intergenic non-coding RNA-RoR modulates reprogramming of human induced pluripotent stem cells.
2010,
Pubmed Luo,
Critical developmental stages for the efficiency of somatic cell nuclear transfer in zebrafish.
2011,
Pubmed Luo,
Identification of differentially expressed genes between cloned and zygote-developing zebrafish (Danio rerio) embryos at the dome stage using suppression subtractive hybridization.
2009,
Pubmed Ma,
Abnormalities in human pluripotent cells due to reprogramming mechanisms.
2014,
Pubmed Mahmoudi,
Wrap53, a natural p53 antisense transcript required for p53 induction upon DNA damage.
2009,
Pubmed Mercer,
Long non-coding RNAs: insights into functions.
2009,
Pubmed Onishi,
Pig cloning by microinjection of fetal fibroblast nuclei.
2000,
Pubmed Pauli,
Systematic identification of long noncoding RNAs expressed during zebrafish embryogenesis.
2012,
Pubmed Ponting,
Evolution and functions of long noncoding RNAs.
2009,
Pubmed Rideout,
Nuclear cloning and epigenetic reprogramming of the genome.
2001,
Pubmed Saunders,
Concise review: pursuing self-renewal and pluripotency with the stem cell factor Nanog.
2013,
Pubmed Seoane,
Myc suppression of the p21(Cip1) Cdk inhibitor influences the outcome of the p53 response to DNA damage.
2002,
Pubmed Serrano,
Gata4 blocks somatic cell reprogramming by directly repressing Nanog.
2013,
Pubmed Silva,
Nanog is the gateway to the pluripotent ground state.
2009,
Pubmed Smith,
Human family with sequence similarity 60 member A (FAM60A) protein: a new subunit of the Sin3 deacetylase complex.
2012,
Pubmed Thisse,
High-resolution in situ hybridization to whole-mount zebrafish embryos.
2008,
Pubmed Wakayama,
Production of cloned mice and ES cells from adult somatic cells by nuclear transfer: how to improve cloning efficiency?
2007,
Pubmed Wakayama,
Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei.
1998,
Pubmed Wani,
Production of the first cloned camel by somatic cell nuclear transfer.
2010,
Pubmed Wilmut,
Viable offspring derived from fetal and adult mammalian cells.
1997,
Pubmed Woods,
A mule cloned from fetal cells by nuclear transfer.
2003,
Pubmed Zhang,
The gut-enriched Kruppel-like factor (Kruppel-like factor 4) mediates the transactivating effect of p53 on the p21WAF1/Cip1 promoter.
2000,
Pubmed Zindy,
Myc signaling via the ARF tumor suppressor regulates p53-dependent apoptosis and immortalization.
1998,
Pubmed
