CN106188272A - 基因工程鼠提高Btg2表达水平的新功能 - Google Patents

基因工程鼠提高Btg2表达水平的新功能 Download PDF

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CN106188272A
CN106188272A CN201610595194.3A CN201610595194A CN106188272A CN 106188272 A CN106188272 A CN 106188272A CN 201610595194 A CN201610595194 A CN 201610595194A CN 106188272 A CN106188272 A CN 106188272A
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genetic engineering
btg2
dnmt1
new function
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4738—Cell cycle regulated proteins, e.g. cyclin, CDC, INK-CCR
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/02—Breeding vertebrates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/01—Animal expressing industrially exogenous proteins

Abstract

DNMT1是一种DNA甲基化酶，其主要催化DNA的CpG甲基化过程。本申请介绍一种通过敲除DNMT1而得到的基因工程鼠的新功能，通过对基因工程鼠大脑皮层进行提取和蛋白表达分析，我们发现基因工程鼠的Btg2基因的表达量相比于正常小鼠有了显著的提高。Btg2是一种抗增殖蛋白，具有调节胞外信号转导、细胞分化等诸多生理功能。因此，该变化表明Btg2受到DNA甲基化形式的表观遗传学调控作用，敲除鼠在细胞分化等方面可能具有明显异常的表现，证实了DNMT1对神经发育的关键调节作用。

Description

基因工程鼠提高Btg2表达水平的新功能
技术领域
本申请属于转基因生物新功能的技术领域，尤其涉及于一种DNMT1敲除鼠可提高Btg2蛋白表达水平的新功能。
背景技术
DNA甲基转移酶1，即DNMT1，是一种催化DNA胞嘧啶甲基化的关键酶。DNA甲基化是生命体感受外界输入信号、调控结构基因表达的重要修饰手段，是表观遗传学的主要范畴之一。DNMT1在维持DNA甲基化特征及DNA的复制修复方面都有重要的作用。DNMT1作为一种基因表达的调控蛋白，其在不同生理条件下的对靶基因选择的差异最终影响到细胞及组织整体功能的改变。
转基因动物是指通过基因工程的手段使某些基因缺失或增量表达而获得的动物品种，其中转基因小鼠，又称基因工程鼠，在现代神经生物学、病理学、毒理学等学科的研究中发挥着不可替代的作用。基因工程鼠对研究蛋白间的相互作用、调控因子对靶蛋白的作用以及表观遗传学的调控作用均有很大的价值。
Btg2蛋白是一类共调控蛋白，可增强或抑制转录因子的活性，该蛋白在调控细胞周期进程和神经基因表达方面起到重要的作用。Btg2在DG区和室下区的神经再生过程的主要功能是调控其终末端分化；在小脑的早期神经分化过程中，Btg2主要调控颗粒状神经元的迁移和分化。因此，Btg2是神经发育过程中的一种重要的周期调控蛋白，其严谨的表达水平指示着正常的神经发育进程。
发明内容
本申请所用的基因工程鼠，即敲除了DNMT1基因的C57BL/6J小鼠，购自北京唯尚立德生物科技有限公司。用于对照的C57BL/6J小鼠购自安徽医科大学。
敲除了DNMT1基因的C57BL/6J小鼠，即所述的“基因工程鼠”，它的大脑皮层的Btg2蛋白的表达水平提高到正常的对照鼠的1.9倍。
蛋白提取和检测的过程如下：
将培养至1个月的基因工程鼠及对照的C57BL/6J小鼠各6只处死并分别取出其左脑的大脑皮层组织。将上述皮层组织用眼科剪剪成小块后用匀浆机进行匀浆，充分研碎，将组织匀浆化。将匀浆液转入1.5 ml的离心管中，12000 rpm在4oC下离心15 min。离心管中的液体分三层，提取中间无色液相，转入新的离心管中。
将提取的蛋白首先通过本领域常规的BCA法测定其蛋白浓度。随后用等体积的2×的SDS 上样缓冲液混合后进行聚丙烯酰胺凝胶电泳。电泳的条件设定为电压120V，电泳时间1.5 h。随后用硝酸纤维素膜进行转膜，转膜条件为60 V转移2 h。将膜清洗后浸泡入含Btg2抗体的溶液中4oC孵育过夜。随后经多次洗净后使用本领域常用的二抗进行孵育，最后进行化学显影，完成蛋白印迹实验。所获得的显影条带的密度值测定及相互间的比较使用ImageJ软件来完成。
实验组和对照组所获得的Btg2蛋白的表达量的比较如图1所示。
从图1中可以看出，基因工程鼠的Btg2蛋白的表达量是对照鼠的1.9倍。
本申请证实了在小鼠中枢神经系统中DNMT1对Btg2的表达水平有显著的调控作用，表明了DNMT1的缺失导致了包括神经再生调控蛋白的表达异常，证明了DNMT1在神经系统发育中起到不可或缺的调控作用。
附图说明
图1 转基因鼠和对照鼠大脑皮层的Btg2蛋白的表达水平。
Control指对照鼠；DNMT-KO指本申请所述的基因工程鼠。
具体实施方式
实施例1
蛋白提取和检测的过程如下：
将培养至1个月的基因工程鼠及对照的C57BL/6J小鼠各6只处死并分别取出其左脑的大脑皮层组织。将上述皮层组织用眼科剪剪成小块后用匀浆机进行匀浆，充分研碎，将组织匀浆化。将匀浆液转入1.5 ml的离心管中，12000 rpm在4oC下离心15 min。离心管中的液体分三层，提取中间无色液相，转入新的离心管中。
将提取的蛋白首先通过本领域常规的BCA法测定其蛋白浓度。随后用等体积的2×的SDS 上样缓冲液混合后进行聚丙烯酰胺凝胶电泳。电泳的条件设定为电压120V，电泳时间1.5 h。随后用硝酸纤维素膜进行转膜，转膜条件为60 V转移2 h。将膜清洗后浸泡入含Btg2抗体的溶液中4oC孵育过夜。随后经多次洗净后使用本领域常用的二抗进行孵育，最后进行化学显影，完成蛋白印迹实验。所获得的显影条带的密度值测定及相互间的比较使用ImageJ软件来完成。

Claims (4)

1.一种基因工程鼠的新功能，其特征在于，其可引起抗增殖蛋白表达水平的改变。
2.一种基因工程鼠的新功能，其特征在于，其Btg2蛋白的表达水平较之正常小鼠有明显的提高。
3.如权利要求2所述的基因工程鼠的新功能，其特征在于，该基因工程鼠是DNMT1基因敲除后的C57BL/6J小鼠。
4.如权利要求3所述的基因工程鼠的新功能，其特征在于，其Btg2蛋白的表达水平是对照鼠的1.9倍。
CN201610595194.3A 2016-07-27 2016-07-27 基因工程鼠提高Btg2表达水平的新功能 Pending CN106188272A (zh)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106929476A (zh) * 2017-03-29 2017-07-07 大连理工大学 一种去甲基化试剂在人白血病细胞中的应用

Citations (2)

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Publication number Priority date Publication date Assignee Title
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CN103081868A (zh) * 2011-11-03 2013-05-08 财团法人卫生研究院 hSCARB2基因转殖鼠的制造及其做为肠病毒感染动物模式的应用

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020165355A1 (en) * 1997-08-29 2002-11-07 Innogenetics N.V. Methylated, SmD homologous peptides, reactive with the antibodies from sera of living beings affected with systemic lupus erythematosus
CN103081868A (zh) * 2011-11-03 2013-05-08 财团法人卫生研究院 hSCARB2基因转殖鼠的制造及其做为肠病毒感染动物模式的应用

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* Cited by examiner, † Cited by third party
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