斑马鱼tbx2b调控心脏房室间隔发育的功能研究*

tbx2是早期心脏发育的关键基因。为进一步探究其对房室间隔(AVC)发育的影响,研究利用CRISPR/Cas9介导的基因敲除技术,成功构建了斑马鱼tbx2b突变体。通过T7E1酶切对其F₀进行敲除效率检测,结果显示平均敲除效率约为57.5%。F₁进一步筛选获得tbx2b杂合突变体,测序结果显示突变类型为11 bp碱基缺失的移码突变。tbx2b杂合子内交获得纯合子,tbx2b纯合突变体在5 dpf死亡并出现早期心脏环化异常表型。斑马鱼整胚原位杂交实验显示在3 dpf tbx2b纯合突变体中, 心脏腔室分化特异性标志基因nppa、nppb表达上调并异位表达在AVC,而AVC发育关键基因has2的表达消失。高效构建tbx2b突变体并初探其对下游基因的影响,为后续深入研究tbx2b对心脏AVC发育的作用奠定了基础,同时加深了人们对早期心脏调控网络的认识。

tbx2b Affects Atrioventricular Canal Development in Zebrafish

tbx2 plays an important role in heart development. To further investigate the gene function in the atrioventricular canal (AVC) development, tbx2b^-/- mutation zebrafish was successfully established using the CRISPR/ Cas9 mediated gene knockout technique. The knockout efficiency of F₀ was detected by T7E1 assay, and the result showed that the average knockout efficiency was about 57.5%. Sanger sequencing confirmed that tbx2b F₁ mutant had a-11bp-base deletion and caused frameshift mutation. Homozygous mutations were lethal in 5 dpf and early embryos suffered from cardiac looping abnormalities. In situ hybridization in 3 dpf tbx2b^-/- showed ectopic expression of nppa and nppb, which are chamber-specific marker genes, and significantly decreased expression of has2, which is AVC marker gene. tbx2b ^-/- mutant was efficiently constructed and effects on downstream genes were discussed, which laid a foundation for further research on the effect of cardiac AVC development and understanding of early cardiac regulatory networks.