|
|
Utilization of genome editing technology to knock out \kur{dnd1} gene in sturgeons
VU THI, Trang
In this study, for the first time we used CRISPR/Cas9 gene editing technology in sturgeons i.e., sterlets (Acipenser ruthenus). The sequences of sgRNA and primers were designed based on published dnd1 sterlet sequence. Each pair of sgRNA oligos after ligation ready duplex DNA fragment was cloned into vector pX330-U6-Chimeric_BB-CBh-hSpCas9 backbone and thereafter the transformation to competent cells Escherichia coli DH5 was done. The plasmid carried sgRNA was extracted for downstream applications. We diluted extracted plasmid with 10% of 2 M KCl and injection into animal pole of fertilized eggs of sterlets at one to four-cell-stage, 4 hours post fertilization (hpf). At the same time, second microinjection with 2.5% FITC-biotin-dextrans was injected into vegetal pole for labelling PGCs. In the control groups, the eggs were only injected by 2.5% FITC into vegetal pole. PGCs of sterlet were visualized and photographed using a uorescent stereo microscope Leica M165 FC. To confirm the presence or deletion/insertion occurring in the target gene, we used MCE-202 MultiNA microchip electrophoresis system for DNA analysis, in which the targeted gene after amplifying by PCR was analyzed. Mutations in both treated and control embryos of sterlet were further assessed by Sanger sequencing of the PCR product. In present study, we successfully established basic protocols such as preparation of competent cells, construction of vector carrying sgRNA and its transformation into competent cells to carry out the CRISPR/Cas9 technology in sturgeons. Less number of PGCs was observed in embryos that were treated with CRISPR/Cas9; however, sequencing did not provide us a reliable evidence for mutation of the targeted gene probably due to an unspecific PCR. Therefore, more authentication of dnd1 knockout should be done in future by more specific PCR and repeated sequencing.
|
|
|
Endocrine disruptors induce transgenerational alterations of the male reproductive parameters and mirna expression profiles in mouse primordial germ cells
Děd, Lukáš ; Brieno-Enríquez, M.A. ; García-López, J. ; Cárdenas, D.B. ; Guibert, S. ; Hourcade, J.de D. ; Pěknicová, Jana ; Weber, M. ; del Mazo, J.
Primordial germ cells (PGCs) are the embryonic precursors of the germ cell linage, which are restricted to form only sperm and oocytes following their specification from pluripotent cells. PGC precursors are specified in the epiblast around 6.25 days post coitum (dpc), and around 7.25 dpc become identifiable in a 40 cell-cluster. In the present study, we used a mouse model to evaluate the trans-generational (F1-F3) effects of vinclozolin (VCZ) administrated in two doses on male reproductive parameters. We observed decreased fertility rate, higher apoptotic rate and histopathologic alterations in adult testis, PGC number reduction, increments of PGCs apoptosis and changes in PGCs gene expression among all three generations. In the attempt to clarify the trans-generational transmition of the altered phenotypes, we performed the microRNA expression and DNA methylation analysis. We observed the significant alteration in the expression of multiple microRNA and microRNA-regulated genes which are important for PGCs specification, including LIN28, let-7 and BLIMP1. Trans-generational deregulation in the expression of factors involved in the Lin28-let-7-Blimp1 pathway can lead to specific VCZ-induced phenotype observed in our study.
|
guest ::
