CN110564773A - 一种Rag1基因缺陷动物模型的制备方法及其应用 - Google Patents
一种Rag1基因缺陷动物模型的制备方法及其应用 Download PDFInfo
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Abstract
本发明提供了一种制备Rag1基因缺陷动物模型的方法,该方法利用基因编辑技术,将鼠Rag1基因编码区破坏,通过免疫***指标评价和病理组织学检查检测了该模型,证实获得的小鼠模型构建成功。该免疫缺陷模型将助力肿瘤移植、免疫学、炎症等领域的研究。
Description
技术领域
本发明属于动物基因工程和基因遗传修饰领域,具体涉及一种Rag1基因缺陷动物模型的构建方法及其应用。
背景技术
复杂的生物过程通常需要进行体内分析,而人体内生物学的研究受到道德和技术的严重限制,因此越来越需要动物模型来进行人体细胞、组织和器官的体内研究。目前,科学家们已经开发了多种动物模型来克服这些限制,并且现在已经成为人体细胞和组织体内研究的重要工具。
免疫缺陷动物是指由于先天遗传突变或用人工方法造成一种或多种免疫***组成成分缺陷的动物。最具代表性的动物是1962年发现的裸小鼠,近年来随着实验动物学和分子生物学的迅猛发展,一些新的免疫缺陷大、小鼠培育成功,在肿瘤学研究中发挥着越来越重要的地位。
在脊椎动物免疫***中,每种抗体都被定制为攻击一种特定抗原(外来蛋白质和碳水化合物)而不攻击身体本身。人类基因组最多有30,000个基因,但它产生了数百万种不同的抗体,这使它能够对数百万种不同抗原的入侵作出反应。免疫***通过改组、切割和重组几百个基因(VDJ基因)来产生这种多样性的抗体,从而在称为V(D)J重组的过程中产生数百万个排列[1]。Recombination-activating genes(RAGs)酶作为多亚基复合物起作用,以诱导抗原受体编码区段和侧翼重组信号序列(RSS)之间的单个双链DNA(dsDNA)分子的切割,这导致RSS的移除以及VD和J序列的最终绑定。RRAGs蛋白启动V(D)J重组,这种改组在B细胞和T细胞成熟过程中发生,对于前B细胞和前T细胞的成熟是必需的。
目前的研究表明,RAG-1和RAG-2必须以协同方式起作用才能激活V(D)J重组。当分离并转染到成纤维细胞样品中时,显示RAG-1无效地诱导V(D)J基因的重组活性;当RAG-1与RAG-2共转染时,重组频率增加了1000倍[2]。这一发现证明RAG基因不仅可以帮助V(D)J重组,而且可以直接诱导V(D)J基因的重组。
因此,Rag1基因敲除的纯合子小鼠V(D)J重组不能正常进行,T细胞和B细胞无法正常发育成熟。Rag1敲除小鼠没有CD3+或TCRαβ+细胞,胸腺中细胞含量比野生型或杂合子低15-130倍,胸腺细胞是CD8-CD4-且大多为IL2R+,脾脏及骨髓中检测不到IgM或IgD,显示成熟的B细胞完全缺失。与Prkdc缺陷的scid模型相比,Rag1敲除模型的成熟淋巴细胞缺失更彻底,且不会有“泄漏(leak)”。
申请人利用基因编辑技术将鼠Rag1基因编码区exon 2破坏,获得的小鼠模型存在T细胞和B细胞发育障碍。该模型是助力肿瘤移植、免疫学、炎症等领域的研究的理想动物模型。在此过程中,我们构建了针对mouse Rag1基因exon2的sgRNA,我们对于基因编辑技术中用到的各个原件包括sgRNA等进行了充分的优化和调整,保证了采用该技术制备Rag1缺陷基因动物模型的高成功率和高准确率。
发明内容
本发明提供了一种制备Rag1基因缺陷动物模型的方法,其特征在于,利用基因编辑技术将鼠Rag1基因的Exon2破坏或删除以建立Rag1基因缺陷鼠模型,敲除序列如SEQ IDNO:2所示。
优选地,其包括以下步骤:
(1)构建表达针对鼠源Rag1基因exon2的sgRNA的质粒;
(2)利用体外转录技术获得相应sgRNA;
(3)将所述sgRNA以及Cas9mRNA或Cas9蛋白共注射或共电转至鼠受精***质或细胞核中,移植入受体母鼠生产Rag1基因缺陷小鼠模型。
优选地,所述sgRNA包括序列如SEQ ID NO:8-13所示的sgRNA。
优选地,所述方法进一步包括利用引物鉴定F0代基因型的步骤。
优选地,其中鉴定所述F0代基因型使用的引物为:
引物名称 | 引物序列 |
XM709816rag1-del-F1 | CTGGTCTCAGACTTGTCTTGCT(SEQNo.14) |
XM709816rag1-del-R1 | GCTGTAGCTAACAGGTGCAT(SEQNo.15) |
XM709816-WT-F1 | AGGCTTAGACACTTCTGCCG(SEQNo.16) |
XM709816-WT-R1 | CTGAGTTCTCTTGCGACGGT(SEQNo.17) |
。
优选地,所述方法进一步包括将F0代阳性鼠与背景鼠配繁获得F1,对F1代鼠尾进行基因鉴定,在F0小鼠鉴定的基础上增加对引物XM709816rag1-del-F1/XM709816rag1-del-R1引物扩增产物的测序工作。
优选地,其中所述测序使用的引物如下:
引物名称 | 引物序列 |
XM709816-DEL-Seq-R1 | TCATGGCACACAGTGGCATG(SEQNo.18) |
XM709816-DEL-Seq-F1 | TGTCCAGTTAGTGTAATGAGGC(SEQNo.19) |
XM709816-DEL-Seq-F2 | CCATTGTTCCCAGGTAGCTTA(SEQNo.20) |
XM709816-DEL-Seq-R2 | TCCTGTTGCTCTTCTGGCC(SEQNo.21) |
。
优选地,所述方法进一步包括对缺陷小鼠模型进行免疫***指标评价和病理组织学检查。
所述免疫***指标评价以流式细胞分选术检测小鼠免疫指标(T/B/NK细胞),判定小鼠的免疫***指标。
所述病理组织学检查是以采用组织学常规制片技术中最为广泛应用的石蜡切片及HE染色方法观察及判断胸腺、脾脏和腹股沟***组织形态变化。
进一步地,还提供所述方法在制备肿瘤移植、免疫学、炎症领域研究的动物模型的应用。
提供一种特异性靶向或删除鼠Rag1基因exon2的sgRNA,其序列如SEQ ID NO:8-13所示。
本发明具有以下积极效果:
1、基于已有知识及技术将Rag1基因编码区exon2完全敲除,获得的小鼠模型存在T细胞和B细胞发育障碍;无泄漏,B6-Rag1被称为“non-leaky scids”;对DNA损伤不敏感:耐辐射、适用于针对DNA损伤的放疗或化药研究;
2.该免疫缺陷模型将助力肿瘤移植、免疫学、炎症等领域的研究。
3.本发明提供了优化的制备Rag1免疫缺陷动物模型的具体操作方法,该方法中最大程度的优化了sgRNA、载体的制备等步骤,确保了动物模型的成功率。
附图说明
图1为F0鉴定电泳图。1~13是以此编号F0小鼠鼠尾DNA为模板进行PCR;B6为阴性对照,是以C57BL/6小鼠鼠尾DNA为模板进行PCR;N为空白对照,无模板的对照;DL2000条带:2000bp\1000bp\750bp\500bp\250bp\100bp。
图2为肝脏中T/B/NK细胞比例检测。与野生型小鼠相比,B6-Rag1-KO小鼠外周血中无成熟的T、B细胞,NK细胞代偿性升高。
图3为F0鉴定电泳图。1~12是以此编号F0小鼠鼠尾DNA为模板进行PCR;B6为阴性对照,是以C57BL/6小鼠鼠尾DNA为模板进行PCR;N为空白对照,无模板的对照TRANS2K PLUSII条带:8000bp\5000bp\3000bp\2000bp\1000bp\750bp\500bp\250bp\100bp。
图4为F1鉴定电泳图。24#、25#、27#、28#、29#、30#、31#、34#、35#是以此编号F1小鼠鼠尾DNA为模板进行PCR;WT为阴性对照,是以C57BL/6小鼠鼠尾DNA为模板进行PCR;N为空白对照,无模板的对照;M条带:8000bp\5000bp\3000bp\2000bp\1000bp\750bp\500bp\250bp\100bp。
图5是外周血中T/B/NK细胞比例检测。说明:与野生型小鼠相比,B6-Rag1-KO小鼠外周血中无成熟的T、B细胞,NK细胞代偿性升高。
图6为脾脏中T/B/NK细胞比例检测。说明:与野生型小鼠相比,B6-Rag1-KO小鼠脾脏中无成熟的T、B细胞,NK细胞代偿性升高。
图7肝脏中T/B/NK细胞比例检测。说明:与野生型小鼠相比,B6-Rag1-KO小鼠肝脏中无成熟的T、B细胞,NK细胞代偿性升高。
图8为小鼠胸腺组织学检测。说明:取小鼠胸腺组织,制作HE染色切片后观察,与野生型小鼠相比,B6-Rag1-KO雄雌小鼠胸腺体积减小,被膜下可见少量脂肪细胞浸润,皮质、髓质界限模糊,淋巴细胞减少,间质增多,排列疏松,发育不全。注:箭头所示为脂肪细胞浸润,三角所示为皮质区,五角星所示为髓质区。
图9为小鼠脾脏组织学检测。说明:取小鼠脾脏组织,制作HE染色切片后观察,与野生型小鼠相比,B6-Rag1-KO雄雌小鼠基本结构消失,无明显髓区、脾小体,围绕中央动脉的细胞基本是未成熟的淋巴细胞,多核巨细胞增多,部分有出血现象。注:三角所示为脾小体,箭头所示为多核巨细胞。
图10为小鼠腹股沟***组织学检测。说明:取小鼠腹股沟***组织,制作HE染色切片后观察,与野生型小鼠相比,B6-Rag1-KO雄雌小鼠基本结构消失,体积减小,淋巴细胞明显减少,排列疏松,间质增多。注:五角星所示为淋巴细胞,箭头所示为间质。
具体实施方式
下面结合附图对本发明做进一步的详细说明,以令本领域技术人员参照说明书文字能够据以实施。
实施例1:Rag1-KO小鼠模型的建系(失败案例)
利用基因编辑技术将小鼠Rag1基因的Exon2破坏建立Rag1基因缺陷小鼠模型。
(1)确定敲除区域
根据Rag1基因结构域选择破坏Exon2,设计sgRNA序列如表1所示。
表1 sgRNA信息
sgRNA名称 | sgRNA序列(5’→3’) |
S1a | GTAAGGTTTCCCCTCTGAGG(SEQNo.1) |
S1b | CCAGTAGTTCCAGAGAAGCCTGG(SEQNo.2) |
S2a | CTTGACTTCCCATCAGCATGG(SEQNo.3) |
S2b | AGACACTTCTGCCGCATCTGTGG(SEQNo.4) |
sgRNA转录制备方法:以PrimerStar Max体系(表3),sgRNA-F、sgRNA-R为引物,测序正确的puc57-sgRNA质粒(1:30稀释)为模板进行PCR,PCR产物进行纯化,制备sgRNA转录制备模板。使用T7-ShortScript体外转录试剂盒(AM1354)进行sgRNA的转录。
(2)Rag1-KO小鼠模型建立
将得到的sgRNA及Cas9-D10A经胞质注射至0.5天的小鼠受精卵中,移植至0.5天假孕雌鼠体内,等小鼠出生后,经基因鉴定筛选出中靶小鼠(F0)。
F0代基因型鉴定。对获得的F0小鼠的鼠尾基因组DNA使用表2引物进行KO鉴定。
表2 F0鉴定引物
引物名称 | 引物序列 |
Rag1-GT-tF1 | TGTCCCCAAATATTGTCAGG(SEQNo.5) |
Rag1-GT-tR1 | GCACATTGGGCTGATGTCT(SEQNo.6) |
PCR反应体系以及反应条件如下表所示:
表3 PCR反应体系
试剂(TakaraR045) | 体积(μl) | 规格 |
PrimeSTARMaxPremix(2×) | 12.5 | \ |
ddH2O | 9.5 | \ |
Primer | 1 | 10μM |
Primer | 1 | 10μM |
Template | 1 |
表4 PCR反应条件
F0鼠尾DNA鉴定电泳图见图1,1-13#小鼠均为KO阳性小鼠,经PCR产物测序选择4#小鼠与背景鼠配繁获得F1,其基因鉴定产物进行测序,确认Rag1基因Exon2删除14bp(gaagcacagaaggagaagga----3259bp----aaccttacctagaacag)。F1大量扩繁后进行互配,获得纯合子。
(3)Rag1-KO小鼠的免疫***指标评价
建系获得的Rag1-KO小鼠免疫***不健全,会引起小鼠免疫***的紊乱(无发育成熟的T/B细胞,NK细胞代偿性增加等),对于确认品系制作有效性至关重要。以流式细胞分选术检测小鼠免疫指标(主要为T/B/NK细胞),判定小鼠的免疫***指标。
流式检测方法如下:
取材:对8周龄C57BL/6背景鼠和Rag1-KO纯合小鼠取肝脏,置于C型管。
消化:C型管中有预冷的酶消化液3ml(PBS含Ca,Mg+2%CS+10mM HEPES+30ugDNase+1.75mg胶原酶D),置于37℃水浴中消化30min。消化完成的脾脏细胞加300ul 0.1M的EDTA终止消化。1mL滤膜过滤除去未消化完全的组织块,每管加2mL FACSbuffer中和EDTA。脾脏使用RBC室温避光裂解红细胞5min,加FACSbuffer洗涤并重悬,分到流式管中100uL,准备孵育抗体。
抗体孵育:加入CD3,CD4,CD8,CD19,CD335,IgM抗体,冰上避光孵育1h;FACSbuffer洗涤,加入FACS buffer,上机检测。上样前5min添加Sytoxblue(终浓度1:10000稀释),区分死活细胞。
肝脏中免疫细胞分群数据分别如图2所示,与野生型小鼠相比,B6-Rag1-KO小鼠T细胞总数出现恢复且出现分化成熟,而B细胞维持缺陷状态,NK细胞比例出现代偿性增高。与预期无成熟T、B细胞相悖,判定模型制作失败。
实施例2:Rag1-KO小鼠模型的建系
利用基因编辑技术将小鼠Rag1基因的Exon2敲除建立Rag1基因缺陷小鼠模型。C57BL/6小鼠是目前研究比较成熟的小鼠品系,C57BL/6小鼠为背景鼠,成功获得了Rag1-KO小鼠模型。
(1)确定敲除区域
根据Rag1基因结构域选择将Exon2全部敲除,具体敲除序列如SEQ No.7所示。
小鼠Rag1基因Exon2序列SEQ No.7
atggctgcctccttgccgtctaccctgagcttcagttctgcacccgatgaaattcaacacccacaaatcaaattttccgagtggaaatttaagctgtttagggtgagatcctttgaaaaggcacccgaagaagcacagaaggagaaggattcctcagaggggaaaccttacctagaacagtctccagtagttccagagaagcctggtggtcagaactcaattctgactcaacgagcactgaaactccatcctaaattttcaaagaaattccatgctgatgggaagtcaagcgacaaagcagttcaccaagccaggcttagacacttctgccgcatctgtgggaatcgtttcaagagtgacgggcacagccggagatacccagtccacgggcccgtggacgctaaaacccaaagtcttttccgaaagaaggaaaaaagagtcacttcctggccagacctcattgccaggattttccggatcgacgtgaaggcagatgttgactccatccacccgacggaattctgccatgactgttggagcatcatgcacagaaagttcagcagttcccacagtcaggtctacttcccaaggaaagtgaccgtggagtggcacccccacacaccgtcctgtgacatctgttttactgcccatcggggactcaagaggaagagacatcagcccaatgtgcagctcagcaagaaactaaaaactgtgctcaaccacgcgagacgggaccgtcgcaagagaactcaggctagggtcagcagcaaggaagtcctgaagaagatctccaactgcagtaagattcatctcagtaccaagcttcttgccgtggacttcccagcacactttgtgaaatccatctcctgccagatatgcgaacacattctggctgatcccgtggagaccagctgcaagcatctattctgtaggatctgcattctcagatgtctcaaagtcatgggcagctattgtccctcttgccgatatccgtgcttccctactgacctggagagcccagtgaagtcctttctgaacatcttgaattctctcatggtcaagtgtcccgcgcaagattgcaatgaggaagtgagtctggaaaaatataaccaccatgtgtcaagccacaaagaatctaaagagactttggtgcatatcaataaagggggacggcctcgccagcatctcctgtcactgacgagaagggcgcagaaacatcggctgagggagctcaagattcaagtcaaagaatttgctgacaaagaagaaggtggagatgtgaaggctgtctgcttgacattgtttctcctggcactgagggcgaggaatgagcacaggcaagctgatgaattagaggccatcatgcaaggcaggggctccgggcttcaaccagctgtttgcttggccatccgtgtcaataccttcctcagctgtagccaataccataagatgtacaggactgtgaaagctatcactgggaggcagatttttcaacctttgcatgctcttcggaatgccgagaaagtccttctgccaggctaccatccctttgagtggcagcccccactgaagaatgtgtcctccagaactgatgttggaattattgatgggctgtctggacttgcctcctctgtggatgagtacccagtagataccattgcgaagaggttccgctacgactctgctttggtgtctgctttgatggacatggaagaagacatcttggaaggcatgagatcccaagatcttgatgactacctgaatggtcccttcacagtggtggtaaaggagtcttgcgatggaatgggggatgtgagtgagaagcacgggagtgggcccgcagttccagaaaaggccgttcgtttctctttcacagtcatgagaattacgatagagcatggttcacagaacgtgaaggtgtttgaggaacccaagcccaattctgaactgtgttgcaagccgttgtgtcttatgctggcagatgagtctgaccatgagacccttactgctattctaagccccctcattgccgagagggaggccatgaagagcagtgaattaacgctggagatgggaggcatccccaggacttttaaattcatcttcaggggcactggatacgatgaaaaacttgtccgggaagtagaaggcttggaagcttctggctcagtctacatctgtacactctgtgacaccacccgtttggaagcctctcagaatcttgtcttccactccataaccagaagccacgccgagaacctgcagcgctatgaggtctggcggtccaatccgtatcatgagtccgtggaagagctccgggaccgggtgaaaggggtctctgccaaacctttcatcgagacagtcccttccatagatgcgcttcactgtgacattggcaatgcagctgaattctataagattttccagctggagataggggaagtgtataaacatcccaatgcctctaaagaggaaaggaagagatggcaggccacgctggacaaacatctccggaaaaggatgaacttaaaaccaatcatgaggatgaatggcaactttgcccggaagcttatgacccaagagactgtagacgcagtttgtgagttaattccttctgaggagaggcatgaagctctcagggagctcatggacctttacctgaagatgaaacccgtgtggcgctcttcatgtcccgctaaagagtgtccagagtccctctgtcagtacagtttcaactcacagcgtttcgcggaactcctctccaccaagttcaaatatagatacgagggcaaaatcaccaattactttcacaaaaccttggcacatgtccctgaaattattgaaagggatggctctatcggggcctgggcaagtgagggaaatgaatcgggtaacaagctgtttagacggtttcggaaaatgaatgccaggcagtccaagtgctatgagatggaagatgtcctgaaacatcactggctgtatacttcaaaatacctccagaagtttatgaatgctcataacgcgttaaaaagctctgggtttaccatgaactcaaaggagaccttaggggaccctttgggcattgaggactctctggaaagccaagattcaatggagttttaa
(2)Rag1敲除小鼠制备的sgRNA筛选
在敲除区域外侧设计针对鼠源序列的sgRNA。设计并合成识别5’端靶位点和3’端靶位点的sgRNA序列,并构建sgRNA表达载体。两端sgRNA识别位点分别位于小鼠Rag1基因的Intron1和3’UTR上,各sgRNA在Rag1上的靶位点序列如表5所示:
表5 sgRNA信息
sgRNA转录制备方法:以PrimerStar Max体系(表3),sgRNA-F、sgRNA-R为引物,测序正确的puc57-sgRNA质粒(1:30稀释)为模板进行PCR,PCR产物进行纯化,制备sgRNA转录制备模板。使用T7-ShortScript体外转录试剂盒(AM1354)进行sgRNA的转录。
(3)Rag1-KO小鼠模型建立
将得到的sgRNA及Cas9Protein共电转至0.5天的小鼠受精卵中,移植至0.5天假孕雌鼠体内,等小鼠出生后,经基因鉴定筛选出中靶小鼠(F0)。
F0代基因型鉴定。对获得的F0小鼠的鼠尾基因组DNA分别使用两对引物进行KO鉴定和野生型鉴定,引物XM709816rag1-del-F1/XM709816rag1-del-R1分别位于Intron1上游及3’UTR下游区域内,如该对引物扩增产生2000bp以内的PCR产物,说明目标sgRNA将小鼠Rag1基因Exon2完全删除;XM709816-WT-F1/XM709816-WT-R1位于小鼠Rag1基因Exon2内,如该对引物扩增产生441bp的PCR产物,说明目标sgRNA未将小鼠Rag1基因Exon2完全删除或未产生删除。
表6 F0鉴定引物
对同一个鼠尾DNA分别采用XM709816rag1-del-F1/XM709816rag1-del-R1引物和XM709816-WT-F1/XM709816-WT-R1引物可分别扩增出2000bp以内和441bp条带小鼠判定为KO杂合阳性F0小鼠。
结果:获得9只KO阳性F0小鼠。如图3所示,F0鼠尾DNA鉴定电泳图,1-5#、8-11#小鼠为KO阳性小鼠。
F0与背景鼠配繁获得F1,对F1代鼠尾进行基因鉴定,在F0小鼠鉴定的基础上增加对引物XM709816rag1-del-F1/XM709816rag1-del-R1引物扩增产物的测序工作,测序引物见表7。F1代小鼠PCR实验结果见图4所示,24#、25#、27#、28#、29#、30#、31#、34#、35#小鼠为KO阳性,测序表明Rag1基因Exon2完全删除(tcttaacttgactcccccccccccc----3259bp----ccaaggggtgacatgttggagtgag),野生型检测也为阳性,表明获得的小鼠为KO杂合小鼠。F1大量扩繁后进行互配,获得纯合子。
表7测序引物
引物名称 | 引物序列 |
XM709816-DEL-Seq-R1 | TCATGGCACACAGTGGCATG(SEQNo.18) |
XM709816-DEL-Seq-F1 | TGTCCAGTTAGTGTAATGAGGC(SEQNo.19) |
XM709816-DEL-Seq-F2 | CCATTGTTCCCAGGTAGCTTA(SEQNo.20) |
XM709816-DEL-Seq-R2 | TCCTGTTGCTCTTCTGGCC(SEQNo.21) |
实施例3:Rag1-KO小鼠模型的检测数据
一、Rag1-KO小鼠的免疫***指标评价
建系获得的Rag1-KO小鼠免疫***不健全,会引起小鼠免疫***的紊乱(无发育成熟的T/B细胞,NK细胞代偿性增加等),对于确认品系制作有效性至关重要。以流式细胞分选术检测小鼠免疫指标(主要为T/B/NK细胞),判定小鼠的免疫***指标。
流式检测方法如下:
取材:对8周龄C57BL/6背景鼠和Rag1-KO纯合小鼠分别取外周血、脾脏、肝脏,置于C型管。
消化:C型管中有预冷的酶消化液3ml(PBS含Ca,Mg+2%CS+10mM HEPES+30ugDNase+1.75mg胶原酶D),置于37℃水浴中消化30min。消化完成的脾脏细胞加300ul 0.1M的EDTA终止消化。1mL滤膜过滤除去未消化完全的组织块,每管加2mL FACSbuffer中和EDTA。脾脏使用RBC室温避光裂解红细胞5min,加FACSbuffer洗涤并重悬,分到流式管中100uL,准备孵育抗体。
抗体孵育:加入CD3,CD4,CD8,CD19,CD335,IgM抗体,冰上避光孵育1h;FACSbuffer洗涤,加入FACS buffer,上机检测。上样前5min添加Sytoxblue(终浓度1:10000稀释),区分死活细胞。
外周血、脾脏和肝脏中免疫细胞分群数据分别如图5、图6、图7所示,与野生型小鼠相比,B6-Rag1-KO小鼠外周血、脾脏和肝脏中无成熟的T、B细胞,NK细胞代偿性升高。
二、Rag1-KO小鼠的病理组织学检查
为进一步评价建系获得的Rag1-KO小鼠,采用组织学常规制片技术中最为广泛应用的石蜡切片及HE染色方法观察及判断胸腺、脾脏和腹股沟***组织形态变化。
石蜡切片方法如下:
固定:10%中性***(即4%中性甲醛)或4%多聚甲醛是病理切片常规使用的固定液,固定液与组织块体积之比大于20:1。固定时,组织块不能贴于壁上。记录固定液的名称、材料的种类及开始固定的时间。其它常用的混合固定液有Bouin氏液、Zenker氏液、FAA液等,应根据需求选择适宜的固定液。
洗涤与脱水:固定后的组织材料需除去残留在组织内的固定液及其结晶沉淀,避免影响以后的染色效果。多数用流水冲洗。乙醇为常用脱水剂,它既能与水相混合,又能与透明剂相混。正丁醇、叔丁醇、丙酮等也可做脱水剂。步骤为梯度乙醇顺序脱水(50%→70%→80%→90%→95%→100%,100%需两到三次)。组织在各级乙醇中的脱水时间视组织块大小而定,一般是1到2小时。
透明:常用的透明剂有二甲苯、苯、氯仿、正丁醇等。各种透明剂均是石蜡的溶剂。一般用1/2乙醇+1/2二甲苯→二甲苯I→二甲苯II,每步约为1小时。透明剂的浸渍时间要根据组织材料块大小及属于囊腔或实质器官而定。如果透明时间过短,则透明不彻底,石蜡难于浸入组织;透明时间过长,则组织硬化变脆,不易切出完整切片。最长为数小时。
浸蜡与包埋:用石蜡取代透明剂,使石蜡浸入组织而起支持作用。通常先把组织材料块放在熔化的石蜡和二甲苯的等量混合液浸渍1~2小时,再先后移入2个熔化的石蜡液中浸渍3小时左右。浸蜡应在高于石蜡熔点3℃左右的温箱中进行,以利石蜡浸入组织内。浸蜡后的组织材料块放在装有蜡液的容器中,根据切片需要调整位置后,放在冷台上冷却,做成含有组织块的蜡块。容器可用光亮且厚的纸折叠成纸盒或金属包埋框盒。如果包埋的组织块数量多,应进行编号,避免弄错。石蜡熔化后应在蜡箱内过滤后lk 58℃或62℃两种,可根据季节及操作环境温度来选用。
切片:包埋好的蜡块用刀片修成规整的四棱台,以少许热蜡液将其底部迅速贴附于小木块上,夹在轮转式切片机的蜡块钳内,使蜡块切面与切片刀刃平行,旋紧。切片刀的锐利程度、蜡块硬度都直接影响切片质量。可用热水或冷水等方法适当改变蜡块硬度。通常切片厚度为4~7微米,切出一片接一片的蜡带,用毛笔轻托轻放在纸上。
贴片与烤片:将展平的蜡片牢附于载玻片上,以免在以后的脱蜡、水化及染色等步骤中二者脱开。将一定长度蜡带(连续切片)或单个蜡片(用刀片断开)于温水(45℃左右)中展平,捞至玻片上铺正,晾干后,将载玻片放入45℃温箱中干燥,也可在37℃温箱中干燥,但需适当延长时间。
切片脱蜡及水化:干燥后的切片需脱蜡及水化才能在水溶性染液中进行染色。用二甲苯脱蜡,再逐级经梯度乙醇复水,直至蒸馏水。如果染料配制于乙醇中,则将切片移至于近似浓度乙醇时,即可染色。
染色:为了观察组织内部的细微结构,需要对组织进行染色。常用的染色法有苏木精伊红染色法(简称HE)。染色后,细胞核为蓝紫色,细胞质为粉红色。
HE染色方法如下:
切片在二甲苯中脱蜡5~10分钟。
移入二甲苯和乙醇(1∶1)混合液中5分钟左右(如经二次二甲苯脱蜡,此步可略)。
依次移入100%、95%、85%、70%乙醇,各级为2~5分钟。最后经蒸馏水转入染液。
苏木精染液染色20-45秒。
水洗玻片上多余染液,0.5~1%盐酸酒精(70%酒精配制)分色片刻。镜检控制,直至细胞核及核内染色质清晰为止,约10秒。
流水冲洗15~30分钟,或者在碳酸锂饱和液中短时间碱化或蓝化,即细胞核呈蓝色。
蒸馏水短洗。
依次经50%,70%,85%,95%乙醇脱水,各级为1-3分钟。
0.1~0.5%伊红染液染色1~5分钟,若着色困难,可在每100毫升染液中加入1~2滴冰醋酸,易于着色且不易脱色。
依次经95%、100%、100%乙醇脱水,二甲苯透明(2次),各级为2分钟。
封片:擦去切片周围多余的二甲苯,切勿干涸,迅速滴加适量中性树胶,再加盖玻片封固。
胸腺、脾脏和腹股沟***病理组织学检测分别如图8、图9、图10所示,与野生型小鼠相比,均表现出免疫缺陷特征。
尽管本发明的实施方案已公开如上,但其并不仅仅限于说明书和实施方式中所列运用。它完全可以被适用于各种适合本发明的领域。对于熟悉本领域的人员而言,可容易地实现另外的修改。因此在不背离权利要求及等同范围所限定的一般概念下,本发明并不限于特定的细节和这里示出与描述的图例。
序列表
<110> 江苏集萃药康生物科技有限公司
<120> 一种Rag1基因缺陷动物模型的制备方法及其应用
<160> 21
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> homo sapiens
<400> 1
gtaaggtttc ccctctgagg 20
<210> 2
<211> 23
<212> DNA
<213> homo sapiens
<400> 2
ccagtagttc cagagaagcc tgg 23
<210> 3
<211> 21
<212> DNA
<213> homo sapiens
<400> 3
cttgacttcc catcagcatg g 21
<210> 4
<211> 23
<212> DNA
<213> homo sapiens
<400> 4
agacacttct gccgcatctg tgg 23
<210> 5
<211> 20
<212> DNA
<213> homo sapiens
<400> 5
tgtccccaaa tattgtcagg 20
<210> 6
<211> 19
<212> DNA
<213> homo sapiens
<400> 6
gcacattggg ctgatgtct 19
<210> 7
<211> 3123
<212> DNA
<213> Mus musculus
<400> 7
atggctgcct ccttgccgtc taccctgagc ttcagttctg cacccgatga aattcaacac 60
ccacaaatca aattttccga gtggaaattt aagctgttta gggtgagatc ctttgaaaag 120
gcacccgaag aagcacagaa ggagaaggat tcctcagagg ggaaacctta cctagaacag 180
tctccagtag ttccagagaa gcctggtggt cagaactcaa ttctgactca acgagcactg 240
aaactccatc ctaaattttc aaagaaattc catgctgatg ggaagtcaag cgacaaagca 300
gttcaccaag ccaggcttag acacttctgc cgcatctgtg ggaatcgttt caagagtgac 360
gggcacagcc ggagataccc agtccacggg cccgtggacg ctaaaaccca aagtcttttc 420
cgaaagaagg aaaaaagagt cacttcctgg ccagacctca ttgccaggat tttccggatc 480
gacgtgaagg cagatgttga ctccatccac ccgacggaat tctgccatga ctgttggagc 540
atcatgcaca gaaagttcag cagttcccac agtcaggtct acttcccaag gaaagtgacc 600
gtggagtggc acccccacac accgtcctgt gacatctgtt ttactgccca tcggggactc 660
aagaggaaga gacatcagcc caatgtgcag ctcagcaaga aactaaaaac tgtgctcaac 720
cacgcgagac gggaccgtcg caagagaact caggctaggg tcagcagcaa ggaagtcctg 780
aagaagatct ccaactgcag taagattcat ctcagtacca agcttcttgc cgtggacttc 840
ccagcacact ttgtgaaatc catctcctgc cagatatgcg aacacattct ggctgatccc 900
gtggagacca gctgcaagca tctattctgt aggatctgca ttctcagatg tctcaaagtc 960
atgggcagct attgtccctc ttgccgatat ccgtgcttcc ctactgacct ggagagccca 1020
gtgaagtcct ttctgaacat cttgaattct ctcatggtca agtgtcccgc gcaagattgc 1080
aatgaggaag tgagtctgga aaaatataac caccatgtgt caagccacaa agaatctaaa 1140
gagactttgg tgcatatcaa taaaggggga cggcctcgcc agcatctcct gtcactgacg 1200
agaagggcgc agaaacatcg gctgagggag ctcaagattc aagtcaaaga atttgctgac 1260
aaagaagaag gtggagatgt gaaggctgtc tgcttgacat tgtttctcct ggcactgagg 1320
gcgaggaatg agcacaggca agctgatgaa ttagaggcca tcatgcaagg caggggctcc 1380
gggcttcaac cagctgtttg cttggccatc cgtgtcaata ccttcctcag ctgtagccaa 1440
taccataaga tgtacaggac tgtgaaagct atcactggga ggcagatttt tcaacctttg 1500
catgctcttc ggaatgccga gaaagtcctt ctgccaggct accatccctt tgagtggcag 1560
cccccactga agaatgtgtc ctccagaact gatgttggaa ttattgatgg gctgtctgga 1620
cttgcctcct ctgtggatga gtacccagta gataccattg cgaagaggtt ccgctacgac 1680
tctgctttgg tgtctgcttt gatggacatg gaagaagaca tcttggaagg catgagatcc 1740
caagatcttg atgactacct gaatggtccc ttcacagtgg tggtaaagga gtcttgcgat 1800
ggaatggggg atgtgagtga gaagcacggg agtgggcccg cagttccaga aaaggccgtt 1860
cgtttctctt tcacagtcat gagaattacg atagagcatg gttcacagaa cgtgaaggtg 1920
tttgaggaac ccaagcccaa ttctgaactg tgttgcaagc cgttgtgtct tatgctggca 1980
gatgagtctg accatgagac ccttactgct attctaagcc ccctcattgc cgagagggag 2040
gccatgaaga gcagtgaatt aacgctggag atgggaggca tccccaggac ttttaaattc 2100
atcttcaggg gcactggata cgatgaaaaa cttgtccggg aagtagaagg cttggaagct 2160
tctggctcag tctacatctg tacactctgt gacaccaccc gtttggaagc ctctcagaat 2220
cttgtcttcc actccataac cagaagccac gccgagaacc tgcagcgcta tgaggtctgg 2280
cggtccaatc cgtatcatga gtccgtggaa gagctccggg accgggtgaa aggggtctct 2340
gccaaacctt tcatcgagac agtcccttcc atagatgcgc ttcactgtga cattggcaat 2400
gcagctgaat tctataagat tttccagctg gagatagggg aagtgtataa acatcccaat 2460
gcctctaaag aggaaaggaa gagatggcag gccacgctgg acaaacatct ccggaaaagg 2520
atgaacttaa aaccaatcat gaggatgaat ggcaactttg cccggaagct tatgacccaa 2580
gagactgtag acgcagtttg tgagttaatt ccttctgagg agaggcatga agctctcagg 2640
gagctcatgg acctttacct gaagatgaaa cccgtgtggc gctcttcatg tcccgctaaa 2700
gagtgtccag agtccctctg tcagtacagt ttcaactcac agcgtttcgc ggaactcctc 2760
tccaccaagt tcaaatatag atacgagggc aaaatcacca attactttca caaaaccttg 2820
gcacatgtcc ctgaaattat tgaaagggat ggctctatcg gggcctgggc aagtgaggga 2880
aatgaatcgg gtaacaagct gtttagacgg tttcggaaaa tgaatgccag gcagtccaag 2940
tgctatgaga tggaagatgt cctgaaacat cactggctgt atacttcaaa atacctccag 3000
aagtttatga atgctcataa cgcgttaaaa agctctgggt ttaccatgaa ctcaaaggag 3060
accttagggg accctttggg cattgaggac tctctggaaa gccaagattc aatggagttt 3120
taa 3123
<210> 8
<211> 22
<212> DNA
<213> homo sapiens
<400> 8
ctcagggtag acggcaagga gg 22
<210> 9
<211> 19
<212> DNA
<213> homo sapiens
<400> 9
ctacctggga acaatgggg 19
<210> 10
<211> 20
<212> DNA
<213> homo sapiens
<400> 10
agcacctaat aggtaccagg 20
<210> 11
<211> 19
<212> DNA
<213> homo sapiens
<400> 11
ggtcttatca cccaagggg 19
<210> 12
<211> 22
<212> DNA
<213> homo sapiens
<400> 12
cacctagcac attgccatgt gg 22
<210> 13
<211> 21
<212> DNA
<213> homo sapiens
<400> 13
caaataccaa cttctatgtg g 21
<210> 14
<211> 22
<212> DNA
<213> homo sapiens
<400> 14
ctggtctcag acttgtcttg ct 22
<210> 15
<211> 20
<212> DNA
<213> homo sapiens
<400> 15
gctgtagcta acaggtgcat 20
<210> 16
<211> 20
<212> DNA
<213> homo sapiens
<400> 16
aggcttagac acttctgccg 20
<210> 17
<211> 20
<212> DNA
<213> homo sapiens
<400> 17
ctgagttctc ttgcgacggt 20
<210> 18
<211> 20
<212> DNA
<213> homo sapiens
<400> 18
tcatggcaca cagtggcatg 20
<210> 19
<211> 22
<212> DNA
<213> homo sapiens
<400> 19
tgtccagtta gtgtaatgag gc 22
<210> 20
<211> 21
<212> DNA
<213> homo sapiens
<400> 20
ccattgttcc caggtagctt a 21
<210> 21
<211> 19
<212> DNA
<213> homo sapiens
<400> 21
tcctgttgct cttctggcc 19
Claims (9)
1.一种制备Rag1基因缺陷动物模型的方法,其特征在于,利用基因编辑技术将鼠Rag1基因的Exon2破坏或删除以建立Rag1基因缺陷鼠模型,敲除序列如SEQ ID NO:2所示。
2.如权利要求1所述的方法,其包括以下步骤:
(1)构建表达针对鼠源Rag1基因exon2的sgRNA质粒,sgRNA序列见下:
(2)利用体外转录技术获得相应sgRNA;
(3)将sgRNA及Cas9 mRNA或Cas9 Protein共注射或共电转至鼠受精***质或细胞核中,并将受精卵移植入受体母鼠生产Rag1基因缺陷小鼠模型。
3.如权利要求1-2任一项所述的方法,进一步包括利用引物鉴定F0代基因型的步骤。
4.如权利要求3所述的方法,其中鉴定所述F0代基因型使用的引物为:
5.如权利要求4所述的方法,进一步包括将F0代阳性鼠与背景鼠配繁获得F1,对F1代鼠尾进行基因鉴定,在F0小鼠鉴定的基础上增加对引物XM709816rag1-del-F1/XM709816rag1-del-R1引物扩增产物的测序工作。
6.如权利要求5所述的方法,其中所述测序使用的引物如下:
。
7.如权利要求6所述的方法,进一步包括对缺陷小鼠模型进行免疫***指标评价和病理组织学检查。
8.权利要求1-7任一项所述的方法在制备肿瘤移植、免疫学、炎症领域研究的动物模型的应用。
9.特异性靶向破坏或删除小鼠Rag1基因exon2的sgRNA,其序列如SEQ ID NO:8-9、11-12所示。
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113106101A (zh) * | 2021-05-11 | 2021-07-13 | 广州欣意生物技术有限公司 | 一种nod遗传背景双基因缺陷小鼠模型的制备方法及应用 |
CN113840918A (zh) * | 2019-03-11 | 2021-12-24 | 莱顿大学医学中心附属莱顿教学医院 | 优化的rag1缺陷基因疗法 |
WO2023224360A1 (ko) * | 2022-05-16 | 2023-11-23 | 서울대학교산학협력단 | 면역 결핍 조류모델 및 그 제조방법 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109777834A (zh) * | 2019-02-25 | 2019-05-21 | 中国科学院广州生物医药与健康研究院 | 一种重度免疫缺陷猪模型及其构建方法和应用 |
CN109868285A (zh) * | 2018-05-29 | 2019-06-11 | 江苏艾尔康生物医药科技有限公司 | 一种免疫缺陷大鼠动物模型的构建方法及应用 |
-
2019
- 2019-08-09 CN CN201910732573.6A patent/CN110564773B/zh active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109868285A (zh) * | 2018-05-29 | 2019-06-11 | 江苏艾尔康生物医药科技有限公司 | 一种免疫缺陷大鼠动物模型的构建方法及应用 |
CN109777834A (zh) * | 2019-02-25 | 2019-05-21 | 中国科学院广州生物医药与健康研究院 | 一种重度免疫缺陷猪模型及其构建方法和应用 |
Non-Patent Citations (2)
Title |
---|
GINA B SCOTT ET AL.: "Detecting variable(V),diversity(D) and joining(J) gene segment recombination using a two-colour fluorescence system", 《MOB DNA》 * |
张化浩等: "Chapaev转座子与Transib转座子与RAG1基因起源与进化的关系", 《华中师范大学学报(自然科学版)》 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113840918A (zh) * | 2019-03-11 | 2021-12-24 | 莱顿大学医学中心附属莱顿教学医院 | 优化的rag1缺陷基因疗法 |
CN113106101A (zh) * | 2021-05-11 | 2021-07-13 | 广州欣意生物技术有限公司 | 一种nod遗传背景双基因缺陷小鼠模型的制备方法及应用 |
CN113106101B (zh) * | 2021-05-11 | 2023-04-07 | 广州欣意生物技术有限公司 | 一种nod遗传背景双基因缺陷小鼠模型的制备方法及应用 |
WO2023224360A1 (ko) * | 2022-05-16 | 2023-11-23 | 서울대학교산학협력단 | 면역 결핍 조류모델 및 그 제조방법 |
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