Ribosomes are large RNA and protein complexes that function as the machinery for translation protein synthesis （Boisvert et al., 2007; Ben-Shem et al., 2011： Henras et al., 2015： Kbatter et al., 2015; McCann et al., 2015）. The eukaryotic ribosome is composed of two subunits, the 60S large subunit （LSU） and the 40S small sub- unit （SSU）, which collectively comprise of four different ribosomal RNA （rRNA） species and more than 70 proteins （Ben-Sbem et al., 2011： Henras et al., 2015; Khatter et al., 2015）. The LSU contains the 28S, 5.8S and 5S rRNAs and the SSU contains the 18S rRNA. The assembly of each subunit is initiated in the nucleolus using the respective rRNAs as backbones （Ben-Shem et al., 2011; Henras et al., 2015; Khatter et al., 2015）.
Yong WangQinfang ZhuLing HuangYanqing ZhuJun ChenJinrong PengLi Jan Lo
Expression-independent gene or polyadenylation[poly（A）]trapping is a powerful tool for genome-wide mutagenesis regardless of whether a targeted gene is expressed.Although a number of poly（A）-trap vectors have been developed for the capture and mutation of genes across a vertebrate genome,further efforts are needed to avoid the 3＇-terminal insertion bias and the splice donor（SD） read-through,and to improve the mutagenicity.Here,we present a Sleeping Beauty（SB） transposon-based vector that can overcome these limitations through the inclusion of three functional cassettes required for gene-finding,gene-breaking and large-scale mutagenesis, respectively.The functional cassette contained a reporter/selective marker gene driven by a constitutive promoter in front of a strong SD signal and an AU-rich RNA-destabilizing element（ARE）,which greatly reduced the SD read-through events,except that the internal ribosomal entry site（IRES） element was introduced in front of the SD signal to overcome the phenomenon of 3＇-bias gene trapping.The breaking cassette consisting of an enhanced splicing acceptor（SA）,a poly（A） signal coupled with a transcriptional terminator（TT） effectively disrupted the transcription of trapped genes.Moreover,the Hsp70 promoter from tilapia genome was employed to drive the inducible expression of SB11,which allows the conditional remobilization of a trap insert from a non-coding region.The combination of three cassettes led to effective capture and disruption of endogenous genes in HeLa cells.In addition,the Cre/LoxP system was introduced to delete the Hsp70-SB11 cassette for stabilization of trapped gene interruption and biosafety. Thus,this poly（A）-trap vector is an alternative and effective tool for identification and mutation of endogenous genes in cells and animals.
Conditional expression of a target gene during zebrafish development is a powerful approach to elucidate gene functions. The tetracycline-controlled systems have been successfully used in the modulation of gene expression in mammalian cells, but few lines of zebrafish carrying these systems are currently available. In this study, we had generated a stable transgenic zebrafish line that ubiquitously expressed the second-generation of reverse Tet transactivator （rtTA-M2）. Southern blotting analysis and high-throughput genome sequencing verifed that a single copy of rtTA-M2 gene had stably integrated into the zebrafish genome. After induction with doxycycline （Dox）, a strong green fluorescent protein （GFP） was seen in rtTA-transgenic eggs injected with pTRE--EGFP plasmids. The fluorescent signal gradually decreased after the withdrawal of Dox and disappeared. However, leaky expression of GFP was undetectable before Dox- induction. Additionally, transgenic embryos expressing rtTA-M2 exhibited no obvious defects in morphological phenotypes, hatching behavior and expression patterns of developmental marker genes, suggesting that rtTA-M2 had little effect on the development of transgenic zebrafish. Moreover, expressed Dickkopf-1 （DKK1） in pTRE-DKKl-injected embryos led to alterations in the expression of marker genes associated with Wnt signaling. Thus, this rtTA-transgenic zebrafish can be utilized to dissect functions of genes in a temporal manner.
