CN105063087A - Transgenic method based on RMCE technology - Google Patents

Transgenic method based on RMCE technology Download PDF

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CN105063087A
CN105063087A CN 201510459245 CN201510459245A CN105063087A CN 105063087 A CN105063087 A CN 105063087A CN 201510459245 CN201510459245 CN 201510459245 CN 201510459245 A CN201510459245 A CN 201510459245A CN 105063087 A CN105063087 A CN 105063087A
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cell
es
rmce
technology
method
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CN 201510459245
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Chinese (zh)
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郑敦武
黎妃凤
王正龙
苏翠
俞晓峰
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赛业(苏州)生物科技有限公司
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Abstract

The invention discloses a transgenic method based on the RMCE technology. The transgenic method comprises the following steps: establishing a carrier by taking Rosa26 carrier as a framework and adding with Lox2272 locus or Attp70 locus; after correct sequencing of the carrier, electrotransferring to an ES cell, and screening by adopting a culture medium containing antibiotics, thus obtaining a positive ES cell; verifying again by PCR or Southern Blot; transferring plasmids with Flp recombinase into the positive ES cell, thus obtaining a new positive ES cell; and administrating the positive ES cell after successful verification into the blastaea of a mouse through micro-injection, and then transferring into a surrogate mother mouse, thus obtaining an RMCE tool mouse with a fluorescent mark. The transgenic method is obtained by integrating RMCE and recombinase technology, thus having the advantages of the RMCE technology, and also being capable of realizing fixed-point implantation of genes, and moreover, the the test cycle is short, the efficiency is high, and the toxicity caused by random insertion of the genes is also reduced.

Description

一种基于RMCE技术的转基因方法 A transgenic method of RMCE Technology

技术领域 FIELD

[0001] 本发明属于基因修饰技术领域,具体涉及一种基于RMCE技术的转基因方法。 [0001] The present invention belongs to the field of genetic modification techniques, particularly relates to a method based on RMCE transgenic technology.

背景技术 Background technique

[0002] 现有的转基因技术,多采用显微注射,其具有试验周期短、目的基因片段长度可达100kb、整合效率高等优点,但还是存在诸多不足和缺点,如外源基因是以随机插入的方式进入受体基因组,其整合位点和拷贝数都无法精确掌握,有可能引起基因沉默,更改细胞或组织特异性基因的表达水平,甚至影响整个基因组的表达情况,从而造成宿主优良性状的丢失,甚至有致死性状的出现。 [0002] The conventional transgenic techniques, the use of microinjection, having a short test period, the gene fragment length up to 100KB, the integration of high efficiency, but still many deficiencies and drawbacks, such as the insertion of foreign genes in a random way into the recipient genome, which is the site of integration and copy number can not accurately grasp, may cause gene silencing, change the cell or tissue expression levels of specific genes, and even affect the expression of the entire genome, resulting in a host excellent traits loss, and even death occurs traits.

[0003] 重组酶介导的盒式交换技术(Recombinase Mediated Massette Exchange,RMCE) 与同类方法相比,具有定点、高效、简单而又可能对基因组进行反复修饰等特点,因而在研究基因功能、分析顺式作用成分以及构建各种转基因动物、建立人类疾病动物模型等方面具有很大的应用价值。 [0003] recombinase-mediated cassette exchange technique (Recombinase Mediated Massette Exchange, RMCE) compared with the same method, having a fixed point, highly efficient, simple, and so on may be repeatedly modified genome, thus the study of gene function analysis cis-acting ingredients and build a variety of transgenic animals, the establishment of animal models of human diseases and so has a great value.

发明内容 SUMMARY

[0004] 发明目的:为了克服上述转基因技术的不足和缺点,研制基于RMCE技术的转基因技术,为科学研究和市场应用提供前期基础,本发明所要解决的技术问题是提供了一种基于RMCE技术的转基因方法。 [0004] Object of the invention: In order to overcome the deficiencies and disadvantages of the above transgenic technology, developed based on transgenic technology RMCE technology, to provide an initial basis for scientific research and market application, the present invention is to solve the technical problem of providing a basis RMCE technology transgenic methods.

[0005] 本发明解决了现有转基因技术插入位置及拷贝数不确定、引起基因沉默、更改细胞或组织特异性基因的表达水平,甚至会影响整个基因组的表达情况。 [0005] The present invention solves the prior art transgene and copy number of the insert position uncertainty, resulting in gene silencing, changing the expression level of cell or tissue specific genes, even affect the expression of the entire genome. 本发明可在短时间内、高效率、精确的获得定点插入的转基因鼠。 The present invention in a short time, high efficiency, accurate insertion point to obtain transgenic mice.

[0006] 技术方案:为了解决上述问题,本发明的技术方案是一种基于RMCE技术的转基因方法,包括以下步骤: [0006] Technical Solution: To solve the above problem, the technical solution of the present invention is a method of transgenic RMCE Technology, comprising the steps of:

[0007] 1)构建载体:以Rosa26载体为框架,加上Lox2272位点或Attp70位点、广谱性启动子、焚光蛋白基因、LoxP位点或Attp70位点、Frt位点、筛选标记和DTA终止区域; [0007] 1) Construction of vectors: The vector is Rosa26 frame, with Lox2272 Attp70 site or sites, broad spectrum promoters, light burning gene, the LoxP site or sites Attp70, Frt sites, and selectable marker termination region DTA;

[0008] 2)载体测序正确后,电转到ES细胞中,用含有抗生素的培养基进行筛选,获得阳性ES细胞; After [0008] 2) vector and sequenced correctly, power to ES cells, were screened with medium containing antibiotic, for a positive ES cells;

[0009] 3) PCR 或Southern Blot 再次验证; [0009] 3) PCR or Southern Blot verification again;

[0010] 4)将带有Flp重组酶的质粒转到阳性ES细胞中,获得没有筛选标记的阳性ES细胞; [0010] 4) The plasmid Flp recombinase with the positive ES cells to obtain a positive selection marker no ES cell;

[0011] 5)将验证正确的阳性ES细胞显微注射到小鼠的囊胚中,而后转移至代孕母鼠中, 获得含有荧光标记的RMCE工具鼠。 [0011] 5) to verify that the correct positive ES cells microinjected into blastocysts of mice, and then transferred to foster mothers, the mice obtained RMCE tool containing a fluorescent marker.

[0012] 其中,上述启动子为EF1 a、CAG、CMV或其他所有广谱性表达的强启动子。 [0012] wherein the promoter is EF1 a, a strong promoter CAG, CMV, or the expression of all other broad spectrum.

[0013] 其中,上述荧光蛋白为eGFP、mCherry、RFP、BFP或其他所有发挥追踪作用的蛋白。 [0013] wherein said fluorescent protein is eGFP, mCherry, RFP, BFP, or all other proteins play a role in tracking.

[0014] 其中,上述筛选标记为Neo、Puro或其他所有能达到筛选目的的蛋白。 [0014] wherein the selection marker as Neo, Puro or screening purposes to achieve all other proteins.

[0015] 有益效果:本发明相对于现有技术,具有以下优点: [0015] Advantageous Effects: The present invention relative to the prior art, has the following advantages:

[0016] 1)操作简单、周期短,节省时间和成本; Simple [0016] 1) operation, short cycle, saving time and money;

[0017] 2)只需确定目的基因的序列信息,即可实现基因的定点插入; [0017] 2) determining the sequence of information only the gene of interest, to achieve site-directed insertion of genes;

[0018] 3)减少随机插入引起的非必要的基因沉默; [0018] 3) reduce unnecessary random insertion of the gene silencing induced;

[0019] 4)避免基因突变造成的胚胎过早死亡等。 [0019] 4) to avoid mutations caused by premature fetal death.

[0020] 本发明的转基因技术,是由RMCE与重组酶技术融合而来,其既有RMCE技术的优点,又能实现基因的定点插入,转基因效率高、同时也减少了基因的随机插入带来的毒性。 [0020] Transgenic technology of the present invention, from a fusion with RMCE recombinase technology, the advantages of both techniques RMCE, but also to achieve site-directed insertion of genes, gene transfer efficiency is high, but also reduces the insertion of random genetic brought toxicity.

[0021] 本发明只需要设计简单的载体,即可实现转基因鼠的构建;利用此工具鼠可以在短时间内高效、特异性的完成基因的定点插入,为人类疾病的研究提供理论依据和动物模型。 [0021] The present invention requires only a simple design vector, constructing a gene transfected mouse can be realized; mice using this tool in a short time efficiently, point-specific gene insertion is completed, provides a theoretical basis for the study of animal and human disease model. 另外此发明也可作为临床和医药研究的配套技术,为人类健康保驾护航。 In addition to this invention can also be used as supporting technology and clinical medical research, protect human health.

附图说明 BRIEF DESCRIPTION

[0022] 图1 :实施例1中工具鼠构建的载体骨架; [0022] Figure 1: Example 1 vector backbone murine tool constructed embodiment;

[0023] 图2 :实施例2中工具鼠构建的载体骨架; [0023] FIG. 2: vector backbone Example 2 Construction of murine tool;

[0024] 图3:PCR扩增获得720bp的eGFP片段和1179bp的EF1a片段琼脂糖凝胶电泳图; [0024] FIG. 3: PCR fragment of 720bp was amplified and EF1a eGFP fragment of 1179bp agarose gel electrophoresis;

[0025] 图4 :EF1 a菌落PCR结果琼脂糖凝胶电泳图; [0025] FIG. 4: EF1 a colony PCR agarose gel electrophoresis results of FIG;

[0026] 图5 :eGFP菌落PCR结果琼脂糖凝胶电泳图; [0026] FIG. 5: eGFP Colony PCR results of agarose gel electrophoresis FIG;

[0027] 图6 :5' arm PCR鉴定结果琼脂糖凝胶电泳图; [0027] FIG. 6: 5 'arm PCR identification results of agarose gel electrophoresis FIG;

[0028] 图7 :3' arm PCR鉴定结果琼脂糖凝胶电泳图; [0028] Fig 7: 3 'arm PCR identification results of agarose gel electrophoresis FIG;

[0029] 图8 :焚光显微镜检测ES细胞图片; [0029] FIG. 8: burning light microscopy image of ES cells;

[0030] 图9 :PCR扩增获得1507bp的目的基因片段琼脂糖凝胶电泳图; [0030] FIG 9: PCR amplified gene fragment of 1507bp by agarose gel electrophoresis;

[0031 ] 图10 :菌落PCR扩增获得748bp的片段琼脂糖凝胶电泳图; [0031] FIG. 10: colony PCR amplified fragment by agarose gel electrophoresis to obtain a 748bp;

[0032] 图11 :PCR验证扩增获得659bp的片段琼脂糖凝胶电泳图; [0032] FIG. 11: PCR amplified fragment verification agarose gel electrophoresis of FIG 659bp;

[0033] 图12 :PCR扩增获得672bp CMV片段、720bp的eGFP片段琼脂糖凝胶电泳图; [0033] FIG. 12: PCR amplified fragment 672bp CMV, eGFP fragment of 720bp agarose gel electrophoresis;

[0034] 图13 :CMV菌落PCR获得513bp的扩增片段琼脂糖凝胶电泳图; [0034] FIG. 13: CMV colony PCR amplified fragment was obtained by agarose gel electrophoresis of 513bp;

[0035] 图14 :eGFP菌落PCR获得615bp的扩增片段琼脂糖凝胶电泳图; [0035] FIG. 14: eGFP colony PCR amplified fragment was obtained by agarose gel electrophoresis of 615bp;

[0036] 图15 :5' arm PCR鉴定结果,获得3. lkb的扩增片段琼脂糖凝胶电泳图; [0036] FIG. 15: 5 'arm PCR identification results obtained amplified fragment 3. lkb FIG agarose gel electrophoresis;

[0037] 图16 :3' arm PCR鉴定结果,获得4. 5kb的扩增片段琼脂糖凝胶电泳图; [0037] FIG. 16: 3 'arm PCR identification results obtained amplified fragment by agarose gel electrophoresis of FIG. 4. 5kb;

[0038] 图17 :焚光显微镜检测ES细胞图片; [0038] FIG. 17: burning light microscopy image of ES cells;

[0039] 图18 :PCR扩增获得845bp的目的基因片段琼脂糖凝胶电泳图; [0039] FIG. 18: PCR amplified gene fragment was obtained by agarose gel electrophoresis of 845bp;

[0040] 图19 :菌落PCR扩增获得421bp的片段琼脂糖凝胶电泳图; [0040] FIG. 19: colony PCR amplified fragment in agarose gel electrophoresis FIG 421bp;

[0041] 图20 :PCR验证扩增获得725bp的片段琼脂糖凝胶电泳图; [0041] FIG. 20: PCR amplified fragment verification agarose gel electrophoresis of FIG 725bp;

[0042] 图21 :加上Lox2272 位点、EF1 a、eGFP 基因、LoxP 位点、Frt 位点、Neo 和DTA 终止区域的R〇sa26载体骨架; [0042] FIG. 21: plus Lox2272 site, EF1 a, eGFP gene, LoxP site, Frt site, Neo vector backbone and DTA R〇sa26 termination region;

[0043] 图22 :EF1 a后连入CHRM4基因的外显子序列; [0043] FIG. 22: CHRM4 ligated into the EF1 a gene exon sequence;

[0044] 图23 :加上Attp70位点、CMV、eGFP基因、AttP70位点、Frt位点、Neo和DTA终止区域的Rosa26载体骨架; [0044] FIG. 23: Attp70 plus sites, CMV, eGFP gene, AttP70 site, Frt site, Neo vector backbone and DTA Rosa26 termination region;

[0045] 图24 :CMV启动子之后连入hNQOl的外显子序列; [0045] FIG. 24: CMV promoter even after the hNQOl exon sequences;

[0046] 图25 :常规转基因载体的构建。 [0046] FIG. 25: Construction of a conventional gene transfer vector.

具体实施方式 detailed description

[0047] 下面结合附图对本发明作更进一步的说明。 [0047] DESCRIPTION OF DRAWINGS The invention further.

[0048] 1、实验材料 [0048] 1 Experimental Materials

[0049] 实施例1 : [0049] Example 1:

[0050] 插入基因简介:人类的Cholinergic Receptor,Muscarinic 4 (CHRM4)基因, Ensembl的基因编号为ENSG00000180720,基因编码区全长1437bp ;其功能多样,如参与抑制腺苷酸环化酶、磷酸肌醇的降解等。 [0050] Introduction of the inserted gene: human Cholinergic Receptor, Muscarinic 4 (CHRM4) gene, as numbered Ensembl ENSG00000180720, full-length coding region of 1437bp; its versatility, such as the inhibition of adenylate cyclase involved in phosphoinositide the degradation. 本实施例的EF1 a、eGFP基因、CHRM4基因等所有的DNA模板由苏州金唯智基因公司合成的。 All EF1 a DNA template according to the present embodiment, eGFP gene, CHRM4 genes from Suzhou Goldvision chi gene synthesis company.

[0051] ( -)工具鼠的构建 Build tools mice - [0051] ()

[0052] 1、载体构建:在Rosa26载体骨架上连入加上Lox2272位点、EF1 a、eGFP基因、 LoxP位点、Frt位点、Neo和DTA终止区域。 [0052] 1. Vector construction: in the Rosa26 ligated into the vector backbone sites plus Lox2272, EF1 a, eGFP gene, the LoxP sites, Frt sites, termination area DTA and the Neo. 载体骨架如图21。 21 vector backbone.

[0053] 1)片段的PCR扩增: [0053] a) PCR amplification of a fragment:

[0054] 引入EF1 a、Lox2272位点的引物: [0054] introduced EF1 a, Lox2272 site primer:

[0055] mRosa26-EFla_F/Hpa] [0055] mRosa26-EFla_F / Hpa]

Figure CN105063087AD00051

GGCTCCGGTGCCCGTCAGT GGCTCCGGTGCCCGTCAGT

[0056] mRosa26_EF 1a- R/ AsisI: [0056] mRosa26_EF 1a- R / AsisI:

Figure CN105063087AD00052

TCACGACACCTGAAATGGAAG TCACGACACCTGAAATGGAAG

[0057] 备注:加粗部分为同源臂、灰色部分为Lox2272位点序列 [0057] Note: The bold part homology arm, the gray part Lox2272 site sequence

[0058] 引入eGFP基因、LoxP位点的引物: [0058] introduced the eGFP gene, LoxP site primer:

Figure CN105063087AD00053

TTACTTGTACAGCTCGTCCATGC TTACTTGTACAGCTCGTCCATGC

[0061] 备注:加粗部分为同源臂、灰色部分为LoxP位点序列 [0061] Note: The bold part homology arm, the gray part of the sequence LoxP sites

[0062] EFla或者eGFP基因的PCR扩增体系: [0062] EFla gene or eGFP PCR amplification system:

[0063] [0063]

Figure CN105063087AD00054

[0064] EFla或者eGFP基因的PCR扩增程序: [0064] EFla or PCR eGFP gene amplification program:

[0065] [0065]

Figure CN105063087AD00061

[0066] PCR扩增结果参见图3, PCR扩增获得720bp的eGFP片段、1179bp的EF1 a片段。 [0066] PCR amplification Referring to FIG 3, PCR amplified a 720bp fragment eGFP, EF1 a fragment of 1179bp.

[0067] 2)将获得的片段连接、转化、涂板、挑单克隆,进行菌落PCR验证: [0067] 2) The fragment was ligated, transformed, plated, monoclonal picked, verified by colony PCR:

[0068] 菌落PCR的模板是来源于上述挑选的单菌落。 [0068] Colony PCR template derived from the above-described single colony was picked. 挑选的单菌落混于10 y L的灭菌的蒸馏水中后,取1. 5 yL作为模板。 Single colonies picked after 10 y L mixed in sterile distilled water, 1. 5 yL taken as template.

[0069] EFla的菌检引物: [0069] EFla bacteria sample primer:

[0070] mRosa26-EFla -SF:CCAGCCTGGTCTACACATCAAG [0070] mRosa26-EFla -SF: CCAGCCTGGTCTACACATCAAG

[0071] mRosa26-EFla -SR:ACCACACACGGCACTTACCTG [0071] mRosa26-EFla -SR: ACCACACACGGCACTTACCTG

[0072] eGFP的菌检引物: [0072] eGFP bacteria sample primer:

[0073] mRosa26-eGFP-SF:CCACAACATCGAGGACGGCAG [0073] mRosa26-eGFP-SF: CCACAACATCGAGGACGGCAG

[0074] Neo_R:CTAAAGCGCATGCTCCAGACTG [0074] Neo_R: CTAAAGCGCATGCTCCAGACTG

[0075] 菌落PCR体系: [0075] Colony PCR system:

[0076] [0076]

Figure CN105063087AD00062

[0077] 菌落PCR程序: [0077] Colony PCR program:

[0078] [0078]

Figure CN105063087AD00063

[0079] 菌落PCR结果:EF1 a菌落PCR结果参见图4,获得515bp的扩增片段,eGFP菌落PCR结果参见图5,获得700bp的扩增片段。 [0079] Colony PCR results: EF1 a colony PCR results Referring to Figure 4, the obtained amplified fragment 515bp, eGFP colony PCR results Referring to Figure 5, the obtained amplified fragment 700bp.

[0080] 3)将验证正确的克隆子送交测序 [0080] 3) to verify the correct clones sequenced sent

[0081] 2、将测序正确的载体电转至ES细胞中,用含有新霉素的培养基筛选,获得阳性ES 细胞; [0081] 2, the electric carrier transferred sequenced ES cells screened with medium containing the neomycin obtained positive ES cells;

[0082] 3、PCR再次验证; [0082] 3, PCR re-verification;

[0083] 1)5'armPCR鉴定引物: [0083] 1) 5'armPCR identifying primer:

[0084] Utexas001_KI_Rl:CTTGGCCCGCATTTACAAGACTATC [0084] Utexas001_KI_Rl: CTTGGCCCGCATTTACAAGACTATC

[0085] Utexas001_5PCR_Fl:TCTCGTCGCTGATTGGCTTCTTT [0085] Utexas001_5PCR_Fl: TCTCGTCGCTGATTGGCTTCTTT

[0086] PCR扩增体系: [0086] PCR amplification system:

[0087] [0087]

Figure CN105063087AD00071

[0088] 上述的DNA模板的获取:阳性的ES细胞用细胞裂解液裂解后就直接使用作为模板;阳性的ES细胞加入lmL细胞裂解液,56°C,过夜即可;细胞裂解液的成分如下:50mmol/ L的Tris,PH8.0 ;100mmol/L的EDTA;mmol/L的NaCl;1%SDS;100yL的蛋白酶K(100mg/ mL) 〇 [0088] The DNA template is acquired: positive ES cells after the cells lysed with lysis buffer used directly as a template; lmL positive ES cells cell lysate was added, 56 ° C, overnight can; the following components of the cell lysate : 50mmol / L of Tris, PH8.0; 100mmol / L of EDTA; mmol / L of NaCl; 1% SDS; 100yL proteinase K (100mg / mL) square

[0089] PCR程序: [0089] PCR program:

[0090] [0090]

Figure CN105063087AD00072

[0091] 鉴定结果参见图6, 5'armPCR鉴定结果,获得3. 7kb的扩增片段。 [0091] Referring to FIG authentication result 6, 5'armPCR identification results, the obtained amplified fragment 3. 7kb.

[0092] 2)3'armPCR鉴定引物 [0092] 2) 3'armPCR identifying primer

[0093] NeoPCRF161 • 3:GCTGACCGCTTCCTCGTGCTTTA [0093] NeoPCRF161 • 3: GCTGACCGCTTCCTCGTGCTTTA

[0094] UtexasOO1_3PCR_R2:AAGACACCAGTTTCAGCCCAAGTTC [0094] UtexasOO1_3PCR_R2: AAGACACCAGTTTCAGCCCAAGTTC

[0095] PCR扩增体系 [0095] PCR amplification system

[0096] [0096]

Figure CN105063087AD00073

[0097] [0097]

Figure CN105063087AD00081

[0098] DNA的获取:阳性的ES细胞用细胞裂解液裂解后就直接使用作为模板;阳性的ES 细胞加入lmL细胞裂解液,56°C,过夜即可;细胞裂解液的成分如下: [0098] DNA acquired: positive ES cells after the cells lysed with lysis buffer used directly as a template; lmL positive ES cells cell lysate was added, 56 ° C, overnight can; cell lysates following components:

[0099] 50mmol/L 的Tris,PH 8. 0 ;100mmol/L 的EDTA ;mmol/L 的NaCl ;1 % SDS ;100 y L 的蛋白酶K(100mg/mL)。 [0099] 50mmol / L of Tris, PH 8. 0; 100mmol / L of EDTA; mmol / L of NaCl; 1% SDS; 100 y L proteinase K (100mg / mL).

[0100] PCR 程序: [0100] PCR program:

[0101] [0101]

Figure CN105063087AD00082

[0102] 鉴定结果参见图7, 3' arm PCR鉴定结果,获得4. 8kb的扩增片段。 [0102] Referring to FIG identification results 7, 3 'arm PCR identification results, the obtained amplified fragment 4. 8kb.

[0103] 4、将带有Flp重组酶的质粒转到阳性ES细胞中,获得没有筛选标记的阳性ES细胞,荧光显微镜检测,ES细胞中具有绿色荧光,如图8。 [0103] 4, the Flp recombinase of the plasmid with positive ES cells to obtain a positive selection marker not ES cells, fluorescence microscope, ES cells with green fluorescence, as shown in FIG 8.

[0104] 5、将阳性ES细胞显微注射到小鼠囊胚中,而后转移至代孕母鼠中,获得含有eGFP 的RMCE工具鼠。 [0104] 5, the positive ES cells are microinjected into mouse blastocysts, and then transferred to foster mothers, the mice obtained RMCE tool containing the eGFP.

[0105] (二)转基因小鼠的制作 [0105] (b) production of transgenic mice transfected

[0106] 1、插入载体的构建:在EFla后连入CHRM4基因的外显子序列,如图22。 [0106] 1, inserted into the vector construct: even after the CHRM4 EFla gene exon sequences, as shown in FIG 22.

[0107] CHRM4基因的PCR扩增引物: [0107] CHRM4 PCR gene amplification primer:

[0108] CK070-KI-F:CTTCCATTTCAGGTGTCGTGAGCCACCATGGCCAACTTCACACCTGTC [0108] CK070-KI-F: CTTCCATTTCAGGTGTCGTGAGCCACCATGGCCAACTTCACACCTGTC

[0109] CK070-KI-R:TCTAGTCCGCGGGTGCGATCGCGGTGGCGACCGGTGGATCCCGCC [0109] CK070-KI-R: TCTAGTCCGCGGGTGCGATCGCGGTGGCGACCGGTGGATCCCGCC

[0110] PCR扩增体系: [0110] PCR amplification system:

[0111] [0111]

Figure CN105063087AD00083

[0112] [0112]

Figure CN105063087AD00091

[0113] PCR扩增程序: [0113] PCR amplification program:

[0114] [0114]

Figure CN105063087AD00092

[0115] PCR扩增结果参见图9 :PCR扩增获得1507bp的目的基因片段。 [0115] PCR amplification see FIG. 9: PCR amplified gene fragment of 1507bp.

[0116] 2、将获得的片段连接、转化、涂板、挑单克隆,进行菌落PCR验证。 [0116] 2, the fragment was ligated, transformed, plated, monoclonal picked, verified by colony PCR.

[0117] 菌落PCR的模板是来源于上述挑选的单菌落。 [0117] Colony PCR template derived from the above-described single colony was picked. 挑选的单菌落混于10 yL的灭菌的蒸馏水中后,取1. 5 yL作为模板。 Single colonies were picked after mixed in 10 yL of sterile distilled water, 1. 5 yL taken as template.

[0118] 目的片段菌检引物: [0118] bacteria sample fragment primer:

[0119] KI070-KI-Scr-F:CCGTAATGCAGAAGAAGACCATG [0119] KI070-KI-Scr-F: CCGTAATGCAGAAGAAGACCATG

[0120] pBas i c_Scr-R:CTAAAGCGCATGCTCCAGACTG [0120] pBas i c_Scr-R: CTAAAGCGCATGCTCCAGACTG

[0121] PCR扩增体系: [0121] PCR amplification system:

[0122] [0122]

Figure CN105063087AD00093

[0123] PCR扩增程序: [0123] PCR amplification program:

[0124] [0124]

Figure CN105063087AD00101

[0125] PCR扩增结果参见图10 :菌落PCR扩增获得748bp的片段。 [0125] Referring to Figure 10 PCR amplification: colony PCR amplified fragment obtained is 748bp.

[0126] 3、将构建好的载体与Cre重组酶的mRNA共同注射到工具鼠的受精卵中; [0126] 3, the vector was constructed with mRNA Cre recombinase mice co-injected fertilized egg to the tool;

[0127] 4、PCR再次鉴定,获得阳性的转基因小鼠。 [0127] 4, PCR identification again, to obtain positive transgenic mice.

[0128] PCR 引物: [0128] PCR primer:

[0129] KI070-KI-Scr-F:CCGTAATGCAGAAGAAGACCATG [0129] KI070-KI-Scr-F: CCGTAATGCAGAAGAAGACCATG

[0130] Rosa26-Scr-R:CAGGCTGCAAATCTCAGCAGCT [0130] Rosa26-Scr-R: CAGGCTGCAAATCTCAGCAGCT

[0131] PCR扩增体系: [0131] PCR amplification system:

[0132] [0132]

Figure CN105063087AD00102

[0133] 该模板为C57BL/6NCrl小鼠的鼠爪,裂解后使用;该C57BL/6NCrl小鼠购买于北京维通利华实验动物技术有限公司; [0133] The template for the rat paw C57BL / 6NCrl mice after lysis use; the C57BL / 6NCrl mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd;

[0134] 鼠爪裂解: [0134] paw cracking:

[0135] 1,在含有鼠爪的doff管中加入500yLTail-lysisbuffer,并加入10yL蛋白酶K,混匀后放到55°恒温箱反应过夜。 [0135] 1, containing added paw doff tube 500yLTail-lysisbuffer, and added 10yL proteinase K, 55 ° into the incubator after overnight reaction mix.

[0136] 2, Doff管中加入500 y L酚-氯仿用以抽提蛋白,使蛋白沉淀。 [0136] 2, Doff tube was added 500 y L phenol - chloroform extraction to protein, protein to precipitate.

[0137] 3,将Doff管13000rpm离心10分钟以去除蛋白沉淀。 [0137] 3, the centrifuge tube Doff 13000rpm 10 minutes to remove protein precipitate.

[0138] 4,取上清液约400 y L到另一干净的Doff管中,并加等体积的异丙醇以沉淀DNA。 [0138] 4, the supernatant was about 400 y L Doff to another clean tube and add an equal volume of isopropanol to precipitate the DNA.

[0139] 5,将Doff管13000rpm离心15分钟,倒掉上清。 [0139] 5, the Doff tube was centrifuged 13000rpm for 15 minutes and the supernatant discarded. 6,在Doff管中加入lmL预冷的70%的酒精,13000印111离心10分钟。 6, was added in lmL Doff tube prechilled 70% ethanol, centrifuged at 13,000 printing 11,110 minutes.

[0140] 7,倒掉上清,晾干Doff管,加100 y L灭菌的超纯水溶解DNA ; [0140] 7. Pour off the supernatant, drying tube Doff, DNA was dissolved in deionised water 100 y L sterilized;

[0141] 8,将doff管放入55°恒温箱过夜,使DNA完全溶解,保存到4°C。 [0141] 8, the doff tube in 55 ° oven overnight to completely dissolve the DNA stored to 4 ° C.

[0142] 其中Tail-lysisbuffer配方如下: [0142] Tail-lysisbuffer wherein formula is as follows:

[0143] 1MTris-Hcl(PH= 8.0)lmL、0.5MEDTA(PH= 8.0)2mL、10 %SDS5mL5MNacl 2mL,加水到lOOmL [0143] 1MTris-Hcl (PH = 8.0) lmL, 0.5MEDTA (PH = 8.0) 2mL, 10% SDS5mL5MNacl 2mL, water was added to lOOmL

[0144] 蛋白酶K的配方如下: [0144] Proteinase K in the formula is as follows:

[0145] 蛋白酶K干粉100mg、50%甘油2. 5mL、Tris-Hcl(PH= 7. 5)50yL、0. 1MCacl21mL、 ddH20 1.45mL ; . [0145] Proteinase K powder 100mg, 50% glycerol 2. 5mL, Tris-Hcl (PH = 7. 5) 50yL, 0 1MCacl21mL, ddH20 1.45mL;

[0146] PCR扩增程序: [0146] PCR amplification program:

[0147] [0147]

Figure CN105063087AD00111

[0148] PCR结果参见图11,PCR验证扩增获得659bp的片段。 [0148] PCR results Referring to FIG. 11, PCR verification of the 659bp amplified fragment obtained.

[0149] 实施例2 [0149] Example 2

[0150] 插入基因简介:人类的NQ01基因,Ensembl的基因编号为14910,基因编码区全长825bp ;其编码还原型辅酶/醌氧化还原酶,又称为DT-硫辛酰胺脱氢酶(DT-diaphorase), 是一种黄素酶,NQ01在器官中分布广泛,但在肝脏、肾脏、肠胃道中水平最高。 [0150] Introduction of the inserted gene: human NQ01 gene, as numbered Ensembl 14910, the full-length coding region of 825 bp; encoding the coenzyme / quinone oxidoreductase, also known DT- diaphorase (DT -diaphorase), it is a flavin enzyme, NQ01 widely distributed in organs, but the highest levels in the liver, kidney, gastrointestinal tract. NQ01酶可受到各种化学物的诱导,如多环芳烃、氢醌、丙烯酸盐、花茎甘蓝(十字花科植物提取物)等。 NQ01 enzyme may be induced by a variety of chemicals, such as polycyclic aromatic hydrocarbons, hydroquinone, acrylate, broccoli (cruciferous plant extract) and the like. NQ01酶属于II相代谢酶,在体内与其它I、II相代谢酶一起构成了体内对外源毒性物质的代谢网络,在机体的解毒代谢中发挥重要作用。 NQ01 phase II metabolic enzymes belong to the enzyme, in vivo with other I, II metabolic enzymes constitute a phase vivo metabolic network with exogenous toxic substances, play an important role in the detoxification of the body. NQ01的多态性会降低NQ01酶对化学物质的解毒能力,增加了患血液疾病的风险。 Polymorphism may reduce the NQ01 NQ01 enzyme detoxification of chemicals increases the risk of blood diseases.

[0151 ] 本实施例的CMV、eGFP基因等所有的DNA模板由苏州金唯智基因公司合成的。 [0151] All DNA template present CMV, eGFP genes embodiment by Suzhou intellectualism gene synthesis company.

[0152] (一)工具鼠的构建 Construction of [0152] (a) tool mice

[0153]1、载体构建:在Rosa26载体骨架上连入加上Attp70位点、CMV、eGFP基因、AttP70 位点、Frt位点、Neo和DTA终止区域。 [0153] 1. Vector construction: in the Rosa26 ligated into the vector backbone sites plus Attp70, CMV, eGFP gene, AttP70 site, Frt sites, termination area DTA and the Neo. 载体骨架如图23。 23 vector backbone.

[0154] 1)片段的PCR扩增: [0154] a) PCR amplification of a fragment:

[0155]引入CMV、eGFP位点的引物: [0155] introduction of CMV, eGFP site primer:

[0156] mRosa26-CMV-F/HpaI: [0156] mRosa26-CMV-F / HpaI:

Figure CN105063087AD00112

[0162] 备注:加粗部分为同源臂、灰色部分为Attp位点序列 [0162] Note: The bold part homology arm, the gray part Attp site sequence

[0163] CMV或eGFP基因的PCR扩增体系: [0163] CMV eGFP gene or PCR amplification system:

[0164] [0164]

Figure CN105063087AD00121

[0165] CMV或eGFP基因的PCR扩增程序: [0165] CMV or PCR eGFP gene amplification program:

[0166] [0166]

Figure CN105063087AD00122

[0167] PCR扩增结果参见图12:PCR扩增获得672bpCMV片段、720bp的eGFP片段 [0167] PCR amplification Referring to FIG. 12: PCR fragment amplified 672bpCMV, eGFP fragment of 720bp

[0168] 2)将获得的片段连接、转化、涂板、挑单克隆,进行菌落PCR验证: [0168] 2) The fragment was ligated, transformed, plated, monoclonal picked, verified by colony PCR:

[0169] 菌落PCR的模板是来源于上述挑选的单菌落。 [0169] Colony PCR template derived from the above-described single colony was picked. 挑选的单菌落混于10yL的灭菌的蒸馏水中后,取1.5 yL作为模板。 Single colonies were picked after 10yL mixed in sterile distilled water, 1.5 yL taken as template.

[0170] CMV的菌检引物: [0170] CMV bacteria sample primer:

[0171] mRosa26-CMV-SF:TTGACGTCAATGGAAAGTCC [0171] mRosa26-CMV-SF: TTGACGTCAATGGAAAGTCC

[0172] Neo_R:CTAAAGCGCATGCTCCAGACTG [0172] Neo_R: CTAAAGCGCATGCTCCAGACTG

[0173] eGFP的菌检引物: [0173] eGFP bacteria sample primer:

[0174] mRosa26-EGFP-SF:CCAGCCTGGTCTACACATCAAG [0174] mRosa26-EGFP-SF: CCAGCCTGGTCTACACATCAAG

[0175] mRosa26-EGFP-SR:GCAAAGACCCCAACGAGAAG [0175] mRosa26-EGFP-SR: GCAAAGACCCCAACGAGAAG

[0176] 菌落PCR体系: [0176] Colony PCR system:

[0177] [0177]

Figure CN105063087AD00123

[0178] 菌落PCR程序: [0178] Colony PCR program:

[0179] [0179]

Figure CN105063087AD00131

[0180] 菌落PCR结果:CMV菌落PCR结果参见图13,获得513bp的扩增片段;eGFP菌落PCR结果参见图14,获得615bp的扩增片段。 [0180] Colony PCR results: CMV Colony PCR results Referring to Figure 13, the obtained amplified fragment 513bp; eGFP colony PCR Results Referring to Figure 14, the obtained amplified fragment 615bp.

[0181] 3)将验证正确的克隆子送交测序 [0181] 3) to verify the correct clones sequenced sent

[0182] 2、将测序正确的载体电转至ES细胞中,用含有新霉素的培养基筛选,获得阳性的ES细胞; [0182] 2, the electric carrier transferred sequenced ES cells screened with medium containing the neomycin obtain positive ES cells;

[0183] 3、PCR再次验证 [0183] 3, PCR verification again

[0184] 1)5' arm PCR 鉴定引物: [0184] 1) 5 'arm PCR primers were identified:

[0185] Rosa26_KI_Rl :GGGAAGACAATAGCAGGCATG [0185] Rosa26_KI_Rl: GGGAAGACAATAGCAGGCATG

[0186] Ro sa26_5PCR_F1 : TCTCGTCGCTGATTGGCTTCTTT [0186] Ro sa26_5PCR_F1: TCTCGTCGCTGATTGGCTTCTTT

[0187] 5' arm PCR鉴定结果参见图15,获得3. lkb的扩增片段。 [0187] 5 'arm PCR identification results of Figure 15, the obtained amplified fragment 3. lkb.

[0188] PCR扩增体系 [0188] PCR amplification system

[0189] [0189]

Figure CN105063087AD00132

[0190] DNA的获取:阳性的ES细胞用细胞裂解液裂解后就直接使用作为模板;阳性的ES 细胞加入lmL细胞裂解液,56°C,过夜即可;细胞裂解液的成分如下:50mmol/L的Tris,PH 8. 0 ;100mmol/L 的EDTA ;mmol/L 的NaCl ;1% SDS ;100 y L 的蛋白酶K(100mg/mL)。 [0190] DNA acquired: positive ES cells used directly as a template cell lysate after lysis; lmL positive ES cells are added to the cell lysate, 56 ° C, overnight can; cell lysates following ingredients: 50mmol / L of Tris, PH 8. 0; 100mmol / L of EDTA; mmol / L of NaCl; 1% SDS; 100 y L proteinase K (100mg / mL).

[0191] PCR 程序: [0191] PCR program:

[0192] [0192]

Figure CN105063087AD00141

[0193] 2)3' arm PCR 鉴定引物 [0193] 2) 3 'arm PCR primers identified

[0194] NeoPCRF 161 • 3 :GCTGACCGCTTCCTCGTGCTTTA [0194] NeoPCRF 161 • 3: GCTGACCGCTTCCTCGTGCTTTA

[0195] Rosa26_3PCR_R : CTTCTGTGACCCACGTAAAGC [0195] Rosa26_3PCR_R: CTTCTGTGACCCACGTAAAGC

[0196] 3' arm PCR鉴定结果参见图16,获得4. 5kb的扩增片段。 [0196] 3 'arm PCR identification results Referring to Figure 16, the obtained amplified fragment 4. 5kb.

[0197] PCR扩增体系 [0197] PCR amplification system

[0198] [0198]

[0199] [0199]

Figure CN105063087AD00142

[0200] DNA的获取:阳性的ES细胞用细胞裂解液裂解后就直接使用作为模板;阳性的ES 细胞加入lmL细胞裂解液,56°C,过夜即可;细胞裂解液的成分如下:50mmol/L的Tris,PH 8. 0 ;100mmol/L 的EDTA ;mmol/L 的NaCl ;1% SDS ;100 y L 的蛋白酶K(100mg/mL)。 [0200] DNA acquired: positive ES cells used directly as a template cell lysate after lysis; lmL positive ES cells are added to the cell lysate, 56 ° C, overnight can; cell lysates following ingredients: 50mmol / L of Tris, PH 8. 0; 100mmol / L of EDTA; mmol / L of NaCl; 1% SDS; 100 y L proteinase K (100mg / mL).

[0201] PCR 程序: [0201] PCR program:

[0202] [0202]

Figure CN105063087AD00143

[0203] 4、将带有Flp重组酶的质粒转到阳性ES细胞中,获得没有筛选标记的阳性ES细胞,荧光显微镜检测,ES细胞中具有绿色荧光,如图17。 [0203] 4, the Flp recombinase of the plasmid with positive ES cells to obtain a positive selection marker not ES cells, fluorescence microscope, ES cells with green fluorescence, as shown in FIG 17.

[0204] 5、将阳性ES细胞显微注射到小鼠胚胎中,而后转移至代孕母鼠中,获得含有eGFP 的RMCE工具鼠。 [0204] 5, the positive ES cells are microinjected into mouse embryos, and then transferred to foster mothers, the mice obtained RMCE tool containing the eGFP.

[0205] (二)转基因小鼠的制作 [0205] (b) production of transgenic mice transfected

[0206] 1、插入载体的构建:在CMV启动子之后连入hNQOl的外显子序列,如图24。 [0206] 1, inserted into the vector construct: the exon sequence even after hNQOl promoter CMV, as shown in FIG 24.

[0207] NQ01基因的PCR扩增引物:NQ01基因的DNA模板由苏州金唯智公司合成; PCR [0207] NQ01 gene amplification primer: DNA template synthesized by the Suzhou NQ01 gene Goldvision chi;

[0208] hNQOl-F:GATACCGCGGGCCCGGGCATGGTCGGCAGAAGAGCACTG [0208] hNQOl-F: GATACCGCGGGCCCGGGCATGGTCGGCAGAAGAGCACTG

[0209] hNQO1-R:TGATCAGTTATCTAGAATTCTCATTTTCTAGCTTTGATCTGG [0209] hNQO1-R: TGATCAGTTATCTAGAATTCTCATTTTCTAGCTTTGATCTGG

[0210] PCR扩增体系: [0210] PCR amplification system:

Figure CN105063087AD00151

[0211] [0211]

[0212] [0212]

[0213] PCR扩增程序: [0213] PCR amplification program:

[0214] [0214]

Figure CN105063087AD00152

[0215] PCR扩增结果参见图18 :PCR扩增获得845bp的目的基因片段。 [0215] PCR amplification see Fig 18: PCR amplified gene fragment of 845bp.

[0216] 2、将获得的片段连接、转化、涂板、挑单克隆,进行菌落PCR验证。 [0216] 2, the fragment was ligated, transformed, plated, monoclonal picked, verified by colony PCR.

[0217] 挑取单菌落至10微升水中,取1. 5微升做模板 [0217] Individual colonies were picked into 10 microliters of water, taken as a template 1.5 l

[0218] 目的片段菌检引物: [0218] bacteria sample fragment primer:

[0219] hNQ01-Scr-F:AGTGGCTCCATGTACTCTCTGC [0219] hNQ01-Scr-F: AGTGGCTCCATGTACTCTCTGC

[0220] hNQ01_Scr-R:CTCTACAAATGTGGTATGGCTG [0220] hNQ01_Scr-R: CTCTACAAATGTGGTATGGCTG

[0221] PCR扩增体系: [0221] PCR amplification system:

[0222] [0222]

[0223] [0223]

Figure CN105063087AD00161

[0224] [0224]

[0225] PCR扩增结果参见图19 :菌落PCR扩增获得421bp的片段。 [0225] Referring to Figure 19 PCR amplification: colony PCR amplified fragment obtained is 421bp.

[0226] 3、将构建好的载体与Cre重组酶的mRNA共同注射到工具鼠的受精卵中; [0226] 3, the vector was constructed with mRNA Cre recombinase mice co-injected fertilized egg to the tool;

[0227] 4、PCR再次鉴定,获得阳性的转基因小鼠。 [0227] 4, PCR identification again, to obtain positive transgenic mice.

[0228] PCR引物: [0228] PCR primer:

[0229] hNQ01-Scr-F:AGTGGCTCCATGTACTCTCTGC [0229] hNQ01-Scr-F: AGTGGCTCCATGTACTCTCTGC

[0230] Rosa26-Scr-R:GGTAGAATTTCGACGACCTGCAG [0230] Rosa26-Scr-R: GGTAGAATTTCGACGACCTGCAG

[0231] PCR扩增体系: [0231] PCR amplification system:

[0232] [0232]

Figure CN105063087AD00162

[0233] 该模板为C57BL/6NCrl小鼠的鼠爪,裂解后使用;该C57BL/6NCrl小鼠购买于北京维通利华实验动物技术有限公司;裂解方法同实施例1中裂解方法。 [0233] The template for the rat paw C57BL / 6NCrl mice after lysis use; the C57BL / 6NCrl mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd; cracking process in Example 1 cleaved with the same method.

[0234] PCR扩增程序: [0234] PCR amplification program:

[0235] [0235]

Figure CN105063087AD00171

[0236] PCR扩增结果参见图20 :PCR验证扩增获得725bp的片段。 [0236] PCR amplification see Fig 20: PCR validation of amplified fragment 725bp.

[0237] 对比例: [0237] Comparative Example:

[0238] 常规转基因技术的操作流程: [0238] General operation flow of transgenic technology:

[0239] 1、转基因载体的构建 Construction [0239] 1, a gene transfer vector

[0240] 图25,按照上述载体模式,将目的片段插入转基因载体即可。 [0240] FIG. 25, according to the above carrier pattern, the fragment can be inserted into a gene transfer vector.

[0241] 2、将构建好的载体线性化,经过电泳纯化、凝胶回收及进一步沉淀纯化后用显微注射法将纯化的外源基因注射到受精卵内。 [0241] 2, the linearized vector was constructed, purified after electrophoresis, the gel was further purified by precipitation and recovering purified by microinjection of exogenous genes injected into the fertilized egg.

[0242] 3、将受精卵移植到代孕母鼠中,饲养后鉴定。 [0242] 3, transplanted into fertilized eggs in the foster mothers, identification after feeding.

[0243] 本发明按照实施例1的做法,注射阳性率为4. 42%,按照实施例2的做法,注射阳性率为4. 10%,均值为4. 26%。 [0243] Following the practice of the present invention of Example 1, an injection rate of 4.42% positive, according to the practice of Example 2, the positive rate of injection of 4.10%, a mean of 4.26%. 而按照上述常规的转基因操作方法,注射阳性率仅为1. 21 %,所以该发明技术的转基因效率高。 And in accordance with the above-described conventional method of operation transgenic, positive injection rate was 1.21%, so the high efficiency of the invention, the transgenic technology. 本发明中的注射阳性率=阳性小鼠数/注射胚胎数。 Injection in the present invention, the positive rate = number of positive mice / the number of injected embryos.

[0244] 以上所述仅是本发明的优选实施方式,应当指出:对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。 [0244] The above are only preferred embodiments of the present invention, it should be noted: to those of ordinary skill in the art, in the present invention without departing from the principles of the premise, can make various improvements and modifications, such modifications and modifications should also be regarded as the protection scope of the present invention.

Claims (4)

  1. 1. 一种基于RMCE技术的转基因方法,其特征在于,包括以下步骤: 构建载体:以R〇sa26载体为框架,加上Lox2272位点或Attp70位点、广谱性启动子、荧光蛋白基因、LoxP位点或Attp70位点、Frt位点、筛选标记和DTA终止区域; 载体测序正确后,电转到ES细胞中,用含有抗生素的培养基进行筛选,获得阳性ES细胞; PCR 或Southern Blot 再次验证; 将带有Flp重组酶的质粒转到阳性ES细胞中,获得没有筛选标记的阳性ES细胞; 将验证正确的阳性ES细胞显微注射到小鼠的囊胚中,而后转移至代孕母鼠中,获得含有荧光标记的RMCE工具鼠。 1. A transgenic Based RMCE method, characterized by comprising the steps of: vector: R〇sa26 carrier is in a frame, with Lox2272 Attp70 site or sites, promoters broad spectrum fluorescent protein gene, Attp70 LoxP site or sites, Frt site, selectable marker region, and termination DTA; the sequenced vector electrically to ES cells, were screened with medium containing antibiotic, for a positive ES cells; the PCR or Southern Blot verification again ; plasmid Flp recombinase with the positive ES cells to obtain a positive selection marker no ES cell; and verify proper positive ES cells microinjected into blastocysts of mice, and then transferred to foster mothers in get RMCE tool mouse containing a fluorescent marker.
  2. 2. 根据权利要求1所述的一种基于RMCE技术的转基因方法,其特征在于,所述启动子为EFl a、CAG、CMV或其他所有广谱性表达的强启动子。 According to one of the claims 1 Transgenic Technology RMCE method, wherein said promoter is a strong promoter EFl a CAG CMV promoter or the expression of all other broad spectrum,,.
  3. 3. 根据权利要求1所述的一种基于RMCE技术的转基因方法,其特征在于,所述荧光蛋白为eGFP、mCherry、RFP、BFP或其他所有发挥追踪作用的蛋白。 According to one of the claims 1 Transgenic Technology RMCE method, wherein the fluorescent protein is eGFP, mCherry, RFP, BFP, or all other proteins play a role in tracking.
  4. 4. 根据权利要求1所述的一种基于RMCE技术的转基因方法,其特征在于,所述筛选标记为Neo、Puro或其他所有能达到筛选目的的蛋白。 According to one of the claims 1 Transgenic Technology RMCE method, wherein said selection marker is a Neo, Puro or screening purposes to achieve all other proteins.
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WO1997009439A1 (en) * 1995-09-01 1997-03-13 Genvec, Inc. Methods and vectors for site-specific recombination
CN102851279A (en) * 2012-05-04 2013-01-02 东北农业大学 Pig ROSA26 specific integration site and application thereof

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WO1996040724A1 (en) * 1995-06-07 1996-12-19 Life Technologies, Inc. Recombinational cloning using engineered recombination sites
WO1997009439A1 (en) * 1995-09-01 1997-03-13 Genvec, Inc. Methods and vectors for site-specific recombination
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