Genome-editing In Zebrafish With CRISPR/Cas9 And Initial Exploration On The Second Wave Of Definitive Hematopoiesis In Zebrafish

PartⅠZebrafish (D. rerio) is a popular aquarium fish native in SE of Himalayan region including India, Pakistan and Bangladesh. This fish was applied as a model organism in scientific research since 1970s. In the past three decades, zebrafish is becoming more and more popular as a model animal, due to its small size and large reproducing rate. Passing by the compliment of sequencing of zebrafish genome, it has uncovered that over 70% of human genes have their paralogs in zebrafish genome, which has made zebrafish more of importance as a model vertebrate in functional genome era.To study the function of one very gene, knock-out is an important, even preferably, a method, for people can study or observe the phenotype when this very gene is absent. In recent years, precise genome-editing technique has been developed over several generations. These are ZFNs, TALENs and CRISPR/Cas9. By comparison with the former two, CRISPR/Cas9 is the most popular one due to its easy management and low expenditure. We used this technique to knock-out genes in zebrafish in this experiment.Firstly, we tested the working dosage of this knock-out system using human codon optimized Cas9 mRNA and designing target specific gRNAs, which is synthesized using the reported protocol and materials. We can draw a conclusion that a dosage larger than 300pg per embryo of Cas9 mRNA (abbr. as mRNA in the following words) or 100pg of gRNA can cause severe death. But we cannot detect mutation below a dosage of 150pg of mRNA, so is it below the dosage of gRNA of 50pg per embryo. Due to our anticipation in the Zebrafish Chrl Genes Knock-out Project, it is possible that I collected sufficient samples of CRISPR/Cas9 targeting design and their working condition, containing 4 genes in my task, acta1a, crfbl7, nup133 and taf51. By some simple statistics on the nucleotides in key sites of a target sequence, I found some regulations of the well-working target, and I designed some gRNAs that target some genes-of-interest in our lab, such as cxcl8a, prdx5 and il10, using the former regulations. The results indicated that the designing targets according to the regulations can somehow promote the probability of well working, but the efficiency and working condition are still not considerable. We can draw a suggestion that the binding efficiency of gRNA and specific genomic DNA has correlation not only with the nucleic acid sequence but also can be affected by the secondary or tertiary structure of genomic DNA. Finally, we have obtained the stable mutated lines in genes acta1a and nup133, but there are 5 genes that the F0 generation is in breeding yet, the mutating efficiency and phenotypes is under evaluation.PartⅡAs a new model vertebrate, the transparent appearance in larval stage, the complete in vitro developmental process and convenient genomic manipulation make zebrafish a good one to perform the live imaging and transgen-induced fluorescence labeling and tracing to resolve scientific issues. Based on the known second wave of definitive hematopoiesis in CHT, together with the transgenic line Tg(coro1a:Kaede) and the photoconversible character of Kaede, we labeled HSPCs separately emerged from the AGM region and CHT, and try to find the recordance and difference between the two waves by transient tracing.By the live imaging of eleven HSPCs from CHT, and intermittent tracking of ninty-three HSPCs from AGM region and 127 HSPCs from CHT, we found that the both waves of HSPCs have the complete potential to differentiate to the whole blood cell lineages. But by comparison with AGM, HSPCs from CHT have more declination to stay in the original place. The difference indicated that these two waves of HSPCs may have the different differentiating routes. Because of the deficiency of Kaede labeling of the short time, we cannot make it clear that these two kinds of HSPCs have whether influences when adult and beyond. Together with a transgenic line Tg(kdrl:CreERT2)fb13Tg which we already poccessed, we try to construct a new transgenic line Tg(coro1a:LoxP-BFP-STOP-LoxP-GFP), by merits of Cre/LoxP recombination system, we can label and trace the HSPCs from distinct definitive hematopoietic waves in a more flexible and wide temporal and spatial scope. We have obtained the stable F1 progeny of this transgenic line currently, but the application and relating research need to be done in the future.