斑马鱼心脏发育候选基因Titin-Cap的CRISPR/Cas9打靶有效性分析

CRISPR/Cas9基因打靶技术是近几年发展起来的一种高效率的定向打靶技术,被认为是遗传领域的革命性技术。Titin-Cap基因是本实验室已初步鉴定的斑马鱼心脏发育候选基因,且国内外目前尚无斑马鱼Titin-Cap基因的敲除品系。为了研究Titin-Cap基因在心脏发育过程中的作用机制,我们利用CRISPR/Cas9基因打靶技术建立斑马鱼Titin-Cap基因的敲除品系。测序结果显示,注射了CRISPR/Cas9 gRNA的胚胎出现双峰,说明在打靶位点附近出现了碱基缺失或插入,证明我们设计的gRNA是有效的。对F0代突变体成鱼的筛选中,测序结果同样显示有阳性结果。这些结果说明用CRISPR/Cas9基因打靶技术成功敲除了斑马鱼Titin-Cap基因,获得了Titin-Cap基因敲除的嵌合体斑马鱼。     

基于新的CRISPR/Cas9技术实现斑马鱼LHX9基因的快速编辑及对F0突变体性腺发育的初筛

目的:CRISPR/Cas9系统在斑马鱼的反向遗传学中的到了广泛应用,但突变基因的表型观察往往需要在突变鱼系的F1中进行,费时较长。LHX9作为LIM家族的一种转录因子,在胚胎早期的泌尿生殖嵴中有广泛分布;且LHX9基因敲除的小鼠存在性腺发育不良。本研究拟通过一种新的CRISPR/Cas9基因编辑技术,采用四条sgRNA对LHX9基因进行VASA转基因斑马鱼的基因敲除,以观察该基因缺陷对斑马鱼性腺发育的影响。方法:利用新的CRISPR/Cas9技术,设计四条针对斑马鱼LHX9基因3号外显子的20bp的sgRNA,通过非克隆体外转录得到靶位点的四条sgRNA。将以上靶位点的四条sgRNA与Cas9核酸酶蛋白同时注射入单细胞期的斑马鱼胚胎内,利用PCR鉴定突变型类型和突变比例。通过对LHX9基因突变体的VASA转基因斑马鱼进行荧光观察,发现LHX9基因缺陷的斑马鱼性腺发育的情况。结果:靶向Exon 3的四条sgRNA可成功编辑斑马鱼LHX9基因,敲除效率高达82%,Sanger测序发现产生10种不同的移码突变类型。通过该方法对VASA转基因斑马鱼的LHX9基因进行编辑,发现LHX9基因突变导致dph6的的斑马鱼原始生殖细胞增殖和迁移受到影响。结论:基于4条sgRNA注射的CRISPR/Cas9技术,可以快速地产生具有突变表型的G0斑马鱼,具有应用潜力。LHX9基因敲除导致原始生殖细胞的发育和迁移受到影响,提示该基因参与了斑马鱼早期性腺的发育。     

红斑马鱼体色观察及敲除mitfa基因对其体色发育的影响

斑马鱼(Danio rerio)是一种常见的模式生物,红斑马鱼为小型观赏性鱼类之一.鱼类体色主要是由皮肤或鳞片上的色素细胞的种类和分布决定的.黑色素细胞诱导转录因子(MITF)主要调控动物黑色素细胞发育和分化.本文观察了红斑马鱼成鱼的背鳍、臀鳍、腹鳍、胸鳍色素细胞的组成和分布,跟踪观察了红斑马鱼早期体色发育过程.通过C...     

CRISPR/Cas9技术在斑马鱼基因修饰中的应用

CRISPR/Cas9系统的应用促进了基因编辑技术的快速发展,现已成功地在不同模式生物中实现了高效的基因修饰,包括DNA序列的点突变、大片段删除以及外源基因的定向插入等。现就CRISPR/Cas9系统在斑马鱼模式动物中建立基因敲除和敲入品系的最新研究进展作一综述。     

Fus在斑马鱼生长发育及两性生长异形中的功能

为研究肉瘤融合基因(fus)在鱼类中的功能, 在斑马鱼中利用CRISPR/Cas9基因突变技术使fus基因产生移码突变。fus-/-纯合突变体斑马鱼发育正常并且完全可育, 但体长和体重在幼鱼和成年阶段都小于野生型斑马鱼。此外, 相较于野生型斑马鱼, fus-/-纯合突变体斑马鱼不存在雌鱼身体比例大于雄鱼的性别异形现象。在fus-/-纯合突变体斑马鱼早期幼鱼发育阶段, 一些生长相关基因包括gh1、ghra、igf1、igf2a、stat5.1和socs6的表达水平显著低于野生型斑马鱼但其运动能力并未受到影响。因此, 不同于fus基因在哺乳动物中的功能, 它在斑马鱼中不参与运动神经元的发育, 但在调节鱼类躯体生长发育与两性生长异形方面有着重要的功能。     

利用CRISPR/Cas9技术建立斑马鱼Asb11基因敲除品系

Asb11基因被报道与斑马鱼Notch信号的激活有关,本研究室过去的研究显示该基因在心肌和骨骼肌中特异性表达。因此推测Asb11基因可能是心脏发育相关候选基因。为了阐明Asb11基因在斑马鱼心脏发育过程中的作用,本文利用CRISPR/Cas9打靶技术构建敲除Asb11基因的斑马鱼品系。首先在线分析筛选出Asb11基因最适合的打靶位点,然后PCR扩增出Asb11基因gRNA的双链c DNA,再将Asb11基因的gRNA和Hcas9的mRNA共同注射到斑马鱼胚胎Ⅰ细胞期胚胎中。进行打靶的有效性检测,发现Asb11基因的一号外显子出现了碱基的缺失,表明CRISPR/Cas9系统对Asb11基因的敲除是有效的。对其F0代、F1代、F2代进行筛选,成功获得了Asb11基因敲除的斑马鱼品系,为探究Asb11在心脏发育中的作用奠定了基础。     

CRISPR/Cas基因编辑技术研究进展及其在斑马鱼中的应用

规律成簇间隔的短回文序列(Clustered regularly interspaced short palindromic repeats,CRISPR)是细菌和古菌中的获得性免疫系统,利用该系统能定点进行基因编辑。最近,科学家发现了新的CRISPR-associated (Cas)蛋白,其中由Cas12a介导的基因编辑能显著降低脱靶率。文中对CRISPR/Cas系统的发现历史、组成和分类、工作原理进行概述,并总结了该系统的最新研究进展及在斑马鱼Danio rerio中的应用。     

斑马鱼eomesb基因突变体的构建及其功能分析

Eomesodermin (Eomes)是T-box转录因子基因家族的成员,在脊椎动物胚胎发育、模式形成和形态发生过程中发挥重要作用.斑马鱼(Danio rerio) eomesa基因的一个点突变导致其母源合子突变体胚胎发育延迟而大量死亡,而eomesa合子突变体可正常发育至性成熟,但缺失背鳍.本研究利用CRISPR/Cas9技术构建了斑马鱼eomesa旁系同源基因eomesb的突变体,eomesb纯合突变体不仅可正常发育至性成熟,而且鳍的发育也完全正常.q-PCR和整体原位杂交实验结果显示,eomesa和eomesb在脑部组织具有重叠的表达域,eomesb在中脑的表达略高于eomesa,故推测两基因在脑部可能行使相似的功能,存在一定的功能冗余.对2,4,6 dpf(Days post fertilization)和6 dpf的WT和突变体q-PCR基因表达分析显示,eomesb突变对eomesa的表达没有明显影响.eomesa-/-;eomesb-/-双突变体未出现背鳍缺失以外的附加表型,推测斑马鱼eomesa和eomesb在背鳍发育过程中可能不存在功能冗余,eomesb不参与背鳍的发育过程.此外,整体原位杂交实验结果显示,eomesb突变也不影响其同源基因tbr1a和tbr1b的早期表达.eomesb纯合突变体未观察到明显表型,可能与其影响的功能不引起明显表型有关,也可能与其它基因的遗传补偿效应有关.本研究结果为深入研究斑马鱼eomesb基因的功能提供了实验材料,同时对研究eomesa与eomesb基因功能分化具有参考价值.     

骨形态发生蛋白9基因敲除小鼠的构建

目的运用CRISR/Cas9技术敲除小鼠基因组中Bmp9基因片段,构建Bmp9基因敲除小鼠。方法根据Bmp9基因的外显子序列,设计一段sgRNA并合成。sgRNA体外转录后和Cas9mRNA混合后显微注射受精卵细胞,注射后的受精卵细胞移植至受体动物获得子代小鼠。提取子代小鼠基因组DNA测序鉴定其基因型。基因型鉴定正确的小鼠与野生型交配后筛选纯合子小鼠。同时取纯合子小鼠心脏、肝、脾、肺、肾,匀浆后提取总RNA和总蛋白,通过qPCR、WB和免疫组化检测BMP9在各组织中的表达。结果设计并合成20bp的sgRNA并进行体外转录,显微注射并回植后得到基因突变小鼠,连续交配后得F2代纯合子。测序结果显示,突变小鼠存在两种基因型,一种为5bp缺失突变,另一种为13bp缺失并伴有1bp插入突变。与野生型C57BL/6相比,qPCR、WB和免疫组化结果均表明基因敲除小鼠肝中BMP9表达显著降低。结论利用CRISPR/Cas9技术成功构建出了BMP9基因敲除小鼠。     

CRISPR/Cas9系统在昆虫中的应用

CRISPR/Cas9(clustered regularly interspaced short palindromic repeat/CRISPR-associated nuclease 9)技术是一种RNA引导的基因组靶向编辑技术,能对基因组序列进行精确编辑,在探究基因功能、修复受损基因、沉默有害基因、改良品质性状等方面具有广阔的应用前景。近年来,随着对CRISPR/Cas9系统研究的不断深入和改造,该系统以其操作简易、省时、高效等优点在生物学研究的众多领域中得以推广和应用,特别是在果蝇(Bombyx mori)、家蚕(silkworm)、埃及伊蚊(Aedes aegypti)和蝴蝶(butterfly)等多种昆虫中。本文概述了CRISPR/Cas9的结构、作用原理及发展优化,总结了CRISPR/Cas9导入昆虫的策略和在昆虫中的应用,以及对CRISPR/Cas9系统产生脱靶问题的应对策略,以期对经济昆虫和有益昆虫的分子育种、害虫的生物技术防控等研究提供参考。     

Itch Is Required for Lateral Line Development in Zebrafish

The zebrafish posterior lateral line is formed during early development by the deposition of neuromasts from a migrating primordium. The molecular mechanisms regulating the regional organization and migration of the primordium involve interactions between Fgf and Wnt/-catenin signaling and the establishment of specific cxcr4b and cxcr7b cytokine receptor expression domains. Itch has been identified as a regulator in several different signaling pathways, including Wnt and Cxcr4 signaling. We identified two homologous itch genes in zebrafish, itcha and itchb, with generalized expression patterns. By reducing itchb expression in particular upon morpholino knockdown, we demonstrated the importance of Itch in regulating lateral line development by perturbing the patterns of cxcr4b and cxcr7b expression. Itch knockdown results in a failure to down-regulate Wnt signaling and overexpression of cxcr4b in the primordium, slowing migration of the posterior lateral line primordium and resulting in abnormal development of the lateral line.     

An Assay for Lateral Line Regeneration in Adult Zebrafish

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al.¹⁷ that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.     

Expression of Brain-Derived Neurotrophic Factor and TrkB in the Lateral Line System of Zebrafish During Development

The neuromasts of the lateral line system are regarded as a model to study the mechanisms of hearing, deafness, and ototoxicity. The neurotrophins (NTs), especially brain-derived neurotrophic factor (BDNF), and its signaling receptor TrkB are involved in the development and maintenance of neuromasts. To know the period in which the BDNF/TrkB complex has more effects in the neuromast biology, the age-related changes were studied. Normal zebrafish from 10 to 180 days post-fertilization (dpf), as well as transgenic ET4 zebrafish 10 and 20 dpf, was analyzed using qRT-PCR, western blot, and immunohistochemistry. BDNF and TrkB mRNAs followed a parallel course, peaking at 20 dpf, and thereafter progressively decreased. Specific immunoreactivity for BDNF and TrkB was found co-localized in all hairy cells of neuromasts in 20 and 30 dpf; then, the number of immunoreactive cells decreased, and by 180 dpf BDNF remains restricted to a subpopulation of hairy cells, and TrkB to a few number of sensory and non-sensory cells. At all ages examined, TrkB immunoreactivity was detected in sensory ganglia innervating the neuromasts. The present results demonstrate that there is a parallel time-related decline in the expression of BDNF and TrkB in zebrafish. Also, the patterns of cell expression suggest that autocrine/paracrine mechanisms for this NT system might occur within the neuromasts. Because TrkB in lateral line ganglia did not vary with age, their neurons are potentially capable to respond to BDNF during the entire lifespan of zebrafish.     

Zebrafish Prion Protein PrP2 Controls Collective Migration Process during Lateral Line Sensory System Development

Prion protein is involved in severe neurodegenerative disorders but its physiological role is still in debate due to an absence of major developmental defects in knockout mice. Previous reports in zebrafish indicate that the two prion genes, PrP1 and PrP2, are both involved in several steps of embryonic development thus providing a unique route to discover prion protein function. Here we investigate the role of PrP2 during development of a mechano-sensory system, the posterior lateral line, using morpholino knockdown and PrP2 targeted inactivation. We confirm the efficiency of the translation blocking morpholino at the protein level. Development of the posterior lateral line is altered in PrP2 morphants, including nerve axonal outgrowth and primordium migration defects. Reduced neuromast deposition was observed in PrP2 morphants as well as in PrP2^−/− mutants. Rosette formation defects were observed in PrP2 morphants, strongly suggesting an abnormal primordium organization and reflecting loss of cell cohesion during migration of the primordium. In addition, the adherens junction proteins, E-cadherin and ß-catenin, were mis-localized after reduction of PrP2 expression and thus contribute to the primordium disorganization. Consequently, hair cell differentiation and number were affected and this resulted in reduced functional neuromasts. At later developmental stages, myelination of the posterior lateral line nerve was altered. Altogether, our study reports an essential role of PrP2 in collective migration process of the primordium and in neuromast formation, further implicating a role for prion protein in cell adhesion.     

Proliferative Regeneration of Zebrafish Lateral Line Hair Cells after Different Ototoxic Insults

Sensory hair cells in the zebrafish lateral line regenerate rapidly and completely after damage. Previous studies have used a variety of ototoxins to kill lateral line hair cells to study different phenomena including mechanisms of hair cell death and regeneration. We sought to directly compare these ototoxins to determine if they differentially affected the rate and amount of hair cell replacement. In addition, previous studies have found evidence of proliferative hair cell regeneration in zebrafish, but both proliferation and non-mitotic direct transdifferentiation have been observed during hair cell regeneration in the sensory epithelia of birds and amphibians. We sought to test whether a similar combination of regenerative mechanisms exist in the fish. We analyzed the time course of regeneration after treatment with different ototoxic compounds and also labeled dividing hair cell progenitors. Certain treatments, including cisplatin and higher concentrations of dissolved copper, significantly delayed regeneration by one or more days. However, cisplatin did not block all regeneration as observed previously in the chick basilar papilla. The particular ototoxin did not appear to affect the mechanism of regeneration, as we observed evidence of recent proliferation in the majority of new hair cells in all cases. Inhibiting proliferation with flubendazole blocked the production of new hair cells and prevented the accumulation of additional precursors, indicating that proliferation has a dominant role during regeneration of lateral line hair cells.     

Afferent Neurons of the Zebrafish Lateral Line Are Strict Selectors of Hair-Cell Orientation

Hair cells in the inner ear display a characteristic polarization of their apical stereocilia across the plane of the sensory epithelium. This planar orientation allows coherent transduction of mechanical stimuli because the axis of morphological polarity of the stereocilia corresponds to the direction of excitability of the hair cells. Neuromasts of the lateral line in fishes and amphibians form two intermingled populations of hair cells oriented at 180° relative to each other, however, creating a stimulus-polarity ambiguity. Therefore, it is unknown how these animals resolve the vectorial component of a mechanical stimulus. Using genetic mosaics and live imaging in transgenic zebrafish to visualize hair cells and neurons at single-cell resolution, we show that lateral-line afferents can recognize the planar polarization of hair cells. Each neuron forms synapses with hair cells of identical orientation to divide the neuromast into functional planar-polarity compartments. We also show that afferent neurons are strict selectors of polarity that can re-establish synapses with identically oriented targets during hair-cell regeneration. Our results provide the anatomical bases for the physiological models of signal-polarity resolution by the lateral line.