Generation A Transgenic Zebrafish Line Tg(cyp1a:mCherry) To Conveniently And Sensitively Monitor POPs In The Environment

Persistent organic pollutants(POPs)are resistant to environmental degradation and contribute to a variety of human health problems due to their persistent,bio-accumulative,semi-volatile,high toxicity and long-range transportation behavior.Among POPs,the most abundant and persistent congeners are 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD)and poly aromatic hydrocarbons(PAHs)which are classified as human carcinogens and can cause serious health problems.The environmental pollution becomes more and more seriously which was caused by POPs.It not only affect the quality and safety of agricultural products,especially in aquatic products,but also directly affect drinking water.It is a serious threat to human health and survival.In view of this,it is improtant todevelop a convenient tool for assessing the presence of TCDD and PAHs in the environment.Cytochrome P450 1A(cyp1a)gene which is used as an effective biomarker is up-regulated dramatically in the presence of TCDD and PAHs by activating the aryl hydrocarbon receptor pathway.To develop a convenient bio-monitoring tool for assessing the presence of TCDD and PAHs in the environment,we generated a transgenic zebrafish line Tg(cyp1a:mCherry)with which cyp1 a promoter driven the red fluorescent protein(RFP)expression.Further,we amplified the fluorescence signals by using Gal4/UAS system and TALE-TF system,both of which were driven by cyp1 a promoter.Finally,to generate a zebrafish line whose mCherry gene was knocked into the ORF of the endogenous gene cyp1 a by CRISPR/Cas9 system,we established a method based on in vitro oocyte storage and fertilization(IVF)to improve the efficiencies of Cas9 genome editing and germline transmission.The contents and results are presented as follow:1,The cyp1 a promoter which was cloned by nest-PCR contained complete 8 xenobiotic response elements(XREs),CAAT-signal and TATA-box.To analyze its function,cyp1 a promoters were constructed to perform the luciferase assay.The results of the Dual luciferase reporter assay showed that the promoter was obvirous TCDD dose dependent.2,We generated 5 Tg(cyp1a:mCherry)transgenic zebrafish lines by using I-SceI meganuclease.Among them,line CM2 and CM3 were obvious TCDD dose dependent.To clarify the genetic background,we detected cyp1a:mCherry copy number and insertion sites in the line CM2 and CM3 by absolute quantitative real-time PCR and genome walking assay.The results showed that the CM2 had two copies of cyp1a:mCherry and one was between the gene tspan11 and sema3 aa.The CM3 had just one copy and located in close vicinity to the gene mfng.3,Then,we treated the Tg(cyp1a: mCherry)embryos with different concentrations of TCDD and five US EPA priority PAHs congeners.The results showed that the expression pattern of mCherry induced by TCDD was consistent with the endogenous gene cyp1 a.The red fluorescence signals were mostly located in the liver and intestine region.Besides,the mCherry was also expressed in the head,eyes and skin.The expression of cyp1 a and mCherry were consistent to the exposure concentrations of PAHs and largely induced by TCDD and ?4-ring PAHs.The results showed that the mCherry expression was largely induced by ?4 ring PAHs i.e.BaP,BaA and BghiP.The NaP and Phe revealed weak genetic induction.The ISH results in the current study showed that the cyp1 a was overexpressed in the liver of zebrafish exposed to BaP,BaA and BghiP.Further more,the cyp1 a was also expressed in the blood vessels exposed to BaA and BghiP and in the skin exposed to BaP.Finally,we evaluated the sensitivity of Tg(cyp1a:mCherry)zebrafish toward environmental samples.The results were consistent with our previous findings that highlighted the sensitivity of Tg(cyp1a:gfp)zebrafish exposed to surface water of TGRA31.In this study,the soil and water samples with higher levels of ?4-ring PAHs induced augmented expression level of cyp1a:mCherry and cyp1 a in Tg(cyp1a:mcherry)zebrafish.Therefore,this newly established zebrafish line will provide a convenient and sensitive way for monitoring of TCDD and other persistent organic chemicals in the environment.4,Furthermore,the Luc/Rluc ratio of the 8 XRE simplified cyp1 a promoter was over 6 times and 5.14 times higher than the complete promoter exposed to 0.01 nM TCDD and 1 nM TCDD,respectively.The 8 XRE simplified cyp1 a promoter showed more sensitivity toward TCDD exposure.In the Gal4/UAS system,the transcriptional activity of cyp1 a promoter was significantly improved.The transcriptional activity of both 8 XRE simplified cyp1 a promoter and complete cyp1 a promoter in 8XRE-Gal4/UAS-Luc and cyp1a-Gal4/UAS-Luc were significantly improved by the Gal4/UAS system and with a dose-dependent manner.However,the Luc/Rluc ratio of the two plasmids were also highly efficient induction by Gal4/UAS system even without TCDD exposure and the values were 59.97 and 84.93,respectively.In addition,we examined the transcription activity of 5 randomly selected TALE-VPs and just one can significantly increase the transcriptional activity of the cyp1 a promoter.But in the case of no TCDD exposure,the Luc/Rluc ratio of the cyp1a+GFP/Luc+TALE-VP64 was over 26.44 times higher than the cyp1a+GFP/Luc.The TALE protein maybe toxic and overexpression of it can make the embryos developmental deformity.Therefore,among them,the modified 8XRE promoter maybe the best choice.5,Finally,we reported a method based on in vitro oocyte storage by injecting oocytes in advance and incubating them in oocyte storage medium to significantly improve the efficiencies of genome editing and germline transmission by in vitro fertilization(IVF)in zebrafish.Compared to conventional methods,the prior micro-injection of zebrafish oocytes improved the efficiency of genome editing,especially for the sgRNAs with low targeting efficiency.Through the in vitro oocyte injection,the mutation rates of those 5 genes(mc4r,mpv17,mstna,mc3 r and mrap2b)were higher than normal method(94.4%,88.9%,91.1%,90.0% and 93.3%,respectively).The efficiencies of germline transmission for mc4 r and mpv17 mutations were higher by 96.7% and 91% and the efficiency of gene knock-in was 49.6%.