Generation of knockout rabbits using transcription activator-like effector nucleases

Zinc-finger nucleases and transcription activator-like effector nucleases are novel gene-editing platformscontributing to redefine the boundaries of modern biological research. They are composed of a non-specificcleavage domain and a tailor made DNA-binding module, which enables a broad range of genetic modifications byinducing efficient DNA double-strand breaks at desired loci. Among other remarkable uses, these nucleases havebeen employed to produce gene knockouts in mid-size and large animals, such as rabbits and pigs, respectively.This approach is cost effective, relatively quick, and can produce invaluable models for human disease studies,biotechnology or agricultural purposes. Here we describe a protocol for the efficient generation of knockout rabbitsusing transcription activator-like effector nucleases, and a perspective of the field.

TALENs技术在基因功能研究中的应用

基因敲除技术广泛应用于研究各类细胞和生物的基因功能。转录激活子样效应因子核酸酶技术(TALENs)是近年发展起来的一种新型基因敲除方法,其利用TALEs蛋白核酸结合域氨基酸序列与其靶位点核酸序列之间存在着恒定对应关系,从而组装出能特异性结合任意DNA序列的模块化蛋白。TALENs技术以其快速、高效的优势对生物基因研究领域产生深远影响,为遗传疾病的治疗提供了新的策略。

Highly efficient generation of GGTA1 knockout pigs using a combination of TALEN m RNA and magnetic beads with somatic cell nuclear transfer

The transcription activator-like effector nuclease （TALEN） technique combined with the somatic cel nuclear transfer （SCNT） method has been successfuly applied for creating geneticaly modiifed pigs. However, methods for isolating cels with bialelic indels requires further improvement because of the relatively low enrichment efifciency of mutated somatic cels. Moreover, little is known regarding the off-target effects of the TALEN system and the heredity of TALEN-modiifed pigs. In this study, an efifcient method to increase the enrichment efifciency of TALEN-mediated bialelic knockout （KO） cels was established, and corresponding geneticaly modiifed pigs with the expected genotype were generated whose off-target effect, fertility and heredity characteristics were aslo evaluated. Two TALEN pairs were constructed to target the porcine α-1,3-galactosyltransferase （GGTA1） gene locus. TALEN mRNA was transfected into the ear ifbroblasts folowed by the enrichment of α-Gal nul cels of minipigs using isolectin B4 （IB4） lectin and magnetic beads. A total of 115 cel colonies were formed and validated to beGGTA1 KO cels by sequencing and 10 bialelic KO cel colonies were used as nuclear donors for SCNT. ThirtyGGTA1 bialelic KO piglets were successfuly delivered and grew normaly. Seventeen potential off-target sites were investigated, and no off-target events were detected in the live piglets. To determine the fertility and heredity characteristics of TALEN-modiifed pigs, 10 mature founders were mated with each other and the mutations were determined to be transmitted to the F1 piglets. We established a robust and safe technology for developing geneticaly modiifed pig lines with expected genotypes for agricultural breeding and biomedical application.

Modulation of mitochondrial bioenergetics as a therapeutic strategy in Alzheimer＇s disease

Alzheimer’s disease （AD） is an increasingly pressing worldwide public-health, social, political and economic concern. Despite significant investment in multiple traditional therapeutic strategies that have achieved success in preclinical models addressing the pathological hallmarks of the disease, these efforts have not translated into any effective disease-modifying therapies. This could be because interventions are being tested too late in the disease process. While existing therapies provide symptomatic and clinical benefit, they do not fully address the molecular abnormalities that occur in AD neurons. The pathophysiology of AD is complex; mitochondrial bioenergetic deficits and brain hypometabolism coupled with increased mitochondrial oxidative stress are antecedent and potentially play a causal role in the disease pathogenesis. Dysfunctional mitochondria accumulate from the combination of impaired mitophagy, which can also induce injurious inflammatory responses, and inadequate neuronal mitochondrial biogenesis. Altering the metabolic capacity of the brain by modulating/potentiating its mitochondrial bioenergetics may be a strategy for disease prevention and treatment. We present insights into the mechanisms of mitochondrial dysfunction in AD brain as well as an overview of emerging treatments with the potential to prevent, delay or reverse the neurodegenerative process by targeting mitochondria.

Cautious optimism in anticipation of hepatitis B curative therapies

Despite relative effectiveness of current hepatitis B therapies,there is still no curative agents available.The new emerging approaches hold promise to achieve cure and loss of hepatitis B surface antigen.Studies or clinical trials investigating new therapies remain small and either focus on patients with low viral load and without hepatotoxic injury or patients with hepatitis D co-infection,which makes it challenging to assess their effectiveness and side effect profile in hepatitis B population.

6-磷酸果糖激酶-2／果糖双磷酸酶-2同工酶3基因敲除小鼠模型的构建

目的建立6-磷酸果糖激酶-2／果糖双磷酸酶-2同工酶3（PFKFB3）基因敲除小鼠模型。方法利用类转录激活样效应因子核酸酶（TALEN）技术，通过构建针对PFKFB3基因的TALEN质粒，并将构建好的TALEN质粒在体外反转录为mRNA。利用原核显微注射分别将0．1μl转录后的浓度为50ng／山的mRNA注射到C57BL／6品系小鼠受精卵中，将受精卵回送到ICR代孕母鼠输卵管中，得到F0代基因敲除杂合子小鼠。将FU代饲养至10周龄后合笼，从而构建基因敲除纯合子小鼠。所有子代鼠出生1周后剪尾，提取RNA后反转录，PCR产物作为模板进行基因测序鉴定基因型。结果通过TALEN技术成功构建了f1D代基因敲除杂合子小鼠3只，基因测序结果表明分别于靶基因位置缺失了10、4、1对碱基对；因PFKFB3基因敲除纯合子具有胚胎致死性，6只F1代基因敲除小鼠的基因测序结果显示，针对靶基因，编号4、5的小鼠缺失了1对碱基对，6号小鼠缺失4对碱基对，7、8、9号小鼠缺失10对碱基对。结论成功构建了PFKFB3基因敲除杂合子（＋／-）小鼠．。

新型靶向基因组编辑技术研究进展

传统的靶向基因组编辑技术改造基因效率非常低,严重制约了基础研究和临床应用。因此,新的靶向基因组编辑工具的研究显得非常重要,以此来提高基因原位修复、定点整合及高通量基因敲除的效率。主要论述了近年来发现的新型靶向基因组编辑技术即锌指核酸酶(ZFN)、转录激活子样效应因子核酸酶(TALENs)、规律成簇间隔短回文重复(CRISPR)/Cas系统。从它们的发现、结构和研究进展及应用前景等方面进行了总结;通过比较三者的优缺点,发现规律成簇间隔短回文重复(CRISPRs)具有明显的优点。