WO2008017234A1

WO2008017234A1 - Cell nuclear transfer method

Info

Publication number: WO2008017234A1
Application number: PCT/CN2007/002239
Authority: WO
Inventors: Yitao Zeng; Fei Jiao; Fanyi Zeng; Shuzhen Huang
Original assignee: Shanghai Jiao Tong University Affiliated Children's Hospital; Shanghai Tao Tao Engineering Co., Ltd
Priority date: 2006-08-03
Filing date: 2007-07-23
Publication date: 2008-02-14
Also published as: CN101117633A; CN101117633B

Abstract

A cell nuclear transfer method, which conducted between the non-human mammal's nuclear donor somatic cell and the recipient oocyte possessing the same mitochondria DNA haplotype. The method can remarkably enhance the fusion rate, cleavage rate and blastula formation rate of the reconstructed embryos, therefore remarkably improves the cell nuclear transfer efficiency.

Description

一种细胞核移植方法技术领域 Cell nuclear transplantation method

本发明属于生物工程技术领域，具体地说，是关于一种细胞核移植方法。背景技术 The invention belongs to the field of bioengineering technology, and in particular to a method of nuclear transfer. Background technique

体细胞核移植（somatic cell nuclear transfer, SCNT) 技术已广泛用于克隆动物和转基因动物的生产，多种不同物种的克隆动物相继问世（Chesne P., Adenot PG.， Viglietta C.， et al. Nat Biotechnol. 2002， 20: 366-369; Woods GL" White L., Vanderwall DK" et al. Science 2003, 301 : 1063 ) . 与常规的转基因动物制备方法相比，克隆技术具有独特的优势，这是由于克隆技术将筛选工作提前到了细胞水平，从而大大缩短了优种动物繁殖周期。目前，效率低下是限制该技术的瓶颈。 Somatic cell nuclear transfer (SCNT) technology has been widely used in the production of cloned and transgenic animals, and cloned animals of various species have been introduced (Chesne P., Adenot PG., Viglietta C., et al. Nat). Biotechnol. 2002, 20: 366-369; Woods GL" White L., Vanderwall DK" et al. Science 2003, 301: 1063 ). Compared to conventional transgenic animal preparation methods, cloning technology has unique advantages, this is Because the cloning technology advances the screening work to the cellular level, the breeding cycle of the elite animals is greatly shortened. Currently, low efficiency is a bottleneck that limits the technology.

线粒体（mitochondrion, mt) 不仅是细胞浆中含量最多的细胞器，更重要的是由于线粒体的高突变率，引起不同种群间及同一种群不同个体间线粒体 DNA (mtDNA)序列的差异，表现出不同的单倍型，这种差异可引起生物性状的不同，例如在奶牛中可引起产奶量及生育能力的差异（Tamassia M.， Heyman Y.， Lavergne Y.， et al. Reproduction 2003, 126: 629-637; Sutrno, Cummins JM.， Greeff J., et al. Theriogenology 2002, 57: 1603-1610; Mannen H.， Kojima T" Oyama K.， et al. J. Anim. Sci. 1998, 76: 36-41 ) ₀ Mitochondrion (mt) is not only the most abundant organelle in cytoplasm, but more importantly, due to the high mutation rate of mitochondria, it causes different mitochondrial DNA (mtDNA) sequences among different populations and different individuals in the same population. Haplotypes, this difference can cause differences in biological traits, such as differences in milk production and fertility in dairy cows (Tamassia M., Heyman Y., Lavergne Y., et al. Reproduction 2003, 126: 629) - 637; Sutrno, Cummins JM., Greeff J., et al. Theriogenology 2002, 57: 1603-1610; Mannen H., Kojima T" Oyama K., et al. J. Anim. Sci. 1998, 76: 36 -41 ) ₀

最近几年，动物繁殖领域中***线粒体单倍型日益成为研究热点。传统的单倍型概念是指使用特定限制性内切酶酶切特定片段后产生的特定酶切片段格局。来自体外胚胎生产（IVP )和体细胞核移植的研究均说明体外胚胎的发育明显受母本 mtDNA单倍型影响，并且伴有*** ATP及 mtDNA拷贝数的差异（Tamassia M.， Nuttinck F.， Reynier Ρ,, et al. Biol. Reprod. 2004, 71 : 697-704; Bruggerhoff K., Zakhartchenko V., Wenigerkind H., et al. Biol. Reprod. 2002, 66: 367-373; Hiendleder S.， Prelle K.， Bruggerhoff K., et al. Biol. Reprod. 2004, 70: 1196-1205)。 In recent years, oocyte mitochondrial haplotypes in the field of animal reproduction have increasingly become research hotspots. The traditional concept of haplotype refers to the pattern of specific fragments produced by digestion of a specific fragment with a specific restriction endonuclease. Studies from both in vitro embryonic production (IVP) and somatic cell nuclear transfer indicate that in vitro embryo development is significantly affected by the maternal mtDNA haplotype and is associated with differences in oocyte ATP and mtDNA copy number (Tamassia M., Nuttinck F. Reynier Ρ,, et al. Biol. Reprod. 2004, 71: 697-704; Bruggerhoff K., Zakhartchenko V., Wenigerkind H., et al. Biol. Reprod. 2002, 66: 367-373; Hiendleder S. , Prelle K., Bruggerhoff K., et al. Biol. Reprod. 2004, 70: 1196-1205).

本申请人在其申请号为 200410016219.7 的中国发明专利申请中公开了一种同体细胞核移植方法，该方法是取同一个体的供核体细胞与去核***进行细胞核移植，结果显示克隆囊胚在数量和质量上均有显著提高。发明人由此联想到，目前由常规核移植方法（异体核移植）得到的胚胎，由于含有不同的受体和供体 mtDNA单倍型，这些单倍型可能影响重构胚的发育能力。发明人在前期的研究中发现，某些类型的***有利于克隆胚胎的发育，可能与胞质中某些 mtDNA单倍型更适合胚胎发育有关，其中核编码因子与某些 mtDNA单倍型相兼容可能发挥了重要作用，从而有利于特定线粒体类型的克隆胚胎的成活。发明内容 In the Chinese patent application No. 200410016219.7, the applicant discloses a method for allogeneic cell nuclear transplantation, which comprises taking a nuclear transfer of a donor cell and an enucleated oocyte of the same individual, and the result shows that the cloned blastocyst is obtained. Significantly improved in both quantity and quality. The inventors thus contemplate that embryos currently obtained by conventional nuclear transfer methods (allogeneic nuclear transfer) may affect the developmental capacity of reconstructed embryos due to the different receptor and donor mtDNA haplotypes. In the previous study, the inventors found that certain types of oocytes are beneficial to the development of cloned embryos, and may be related to the development of certain mtDNA haplotypes in the cytoplasm, which are more suitable for embryonic development. The compatibility of mtDNA haplotypes may play an important role in facilitating the survival of cloned embryos of a particular mitochondrial type. Summary of the invention

本发明的目的就在于提供一种细胞核移植方法，该方法选取线粒体 DNA单倍型相同的非人哺乳动物的供核体细胞和受体***进行核移植。 SUMMARY OF THE INVENTION An object of the present invention is to provide a method for nuclear transfer which comprises nuclear transfer of a donor cell and a recipient oocyte of a non-human mammal having the same haplotype DNA haplotype.

为达到上述目的，发明人通过 PCR- RFLP技术对实验动物的线粒体 DNA进行单倍型分析，将不同实验动物的线粒体 DNA单倍型分为 4种类型，然后利用单倍型相同的供核体细胞和受体***进行核移植，结果发现采用单倍型相同的细胞核移植能够有效提高重构胚的发育能力。 In order to achieve the above object, the inventors performed haplotype analysis of mitochondrial DNA of experimental animals by PCR-RFLP technique, and classified mitochondrial DNA haplotypes of different experimental animals into four types, and then utilized the same haplotype nucleus. The nuclear transfer of the cells and the recipient oocytes revealed that the nuclear transfer with the same haplotype can effectively improve the developmental ability of the reconstructed embryo.

具体地，本发明的细胞核移植方法包括以下步骤： Specifically, the nuclear transfer method of the present invention comprises the following steps:

a、选取某一线粒体 DNA单倍型的非人哺乳动物，以其成皮肤成纤维细胞或卵丘细胞作为供核体细胞； a. selecting a non-human mammal of a mitochondrial DNA haplotype, and using it as a dermal fibroblast or a cumulus cell as a donor cell;

b、选取线粒体 DNA单倍型相同的同一种哺乳动物，以其***作为受体细胞； c、受体***去核； b. selecting the same mammal with the same haplotype DNA haplotype, using the oocyte as the recipient cell; c, removing the host oocyte;

d、将供核体细胞的细胞核移入去核***的细胞质中，使细胞核融入***，形成重构胚。 d. The nuclei of the nuclear donor cells are transferred into the cytoplasm of the enucleated oocyte, and the nuclei are integrated into the oocyte to form a reconstructed embryo.

本发明的同型（线粒体 DNA单倍型相同）细胞核移植方法能够有效提高重构胚的融合率、卵裂率以及囊胚率，相比异型（线粒体 DNA单倍型不同）核移植，囊胚率提高了约 1.5倍。 The same type (the same mitochondrial DNA haplotype) cell nuclear transfer method can effectively improve the fusion rate, cleavage rate and blastocyst rate of the reconstructed embryo, compared with the heterotypic (mitochondrial DNA haplotype) nuclear transfer, blastocyst rate Increased by about 1.5 times.

细胞核和细胞质的相互作用和协调是发挥生物功能的基础，在长期的进化过程中，不同的核背景形成了与之相适应的特定单倍型的线粒体 DNA。因此，利用具有相同单倍型的供核细胞和***可有效解决核移植技术中核质间的不兼容，从而提高核移植效率。具体实施方式 ' The interaction and coordination of the nucleus and cytoplasm is the basis of biological function. During the long-term evolution, different nuclear backgrounds form a specific haplotype of mitochondrial DNA. Therefore, the use of donor cells and oocytes with the same haplotype can effectively solve the incompatibility between nuclear materials in nuclear transfer technology, thereby improving nuclear transfer efficiency. detailed description '

以下结合具体实施例对本发明作进一步说明。应理解，以下实施例仅用于说明本发明而不用于限定本发明的范围。 The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the following examples are merely illustrative of the invention and are not intended to limit the scope of the invention.

PCR-RFLP (polymerase chain reaction— restriction fragment length polymorphism)是指采用 PCR技术扩增目的 DNA片段，然后将待检测的片段用限制性内切酶酶切，限制性内切酶识别并切割特异的序列，之后将酶切后的产物进行电泳，根据 DNA片段的大小来比对不同来源基因序列的差异性。本发明就是利用 PCR-RFLP技术，扩增不同牛的线粒体 DNA，然后进行 RPLP分析，选出具有长度差异的片段，根据酶切图谱将其分为 A、 B、 C、 D 四种类型。然后选择线粒体 DNA单倍型相同的细胞进行核移植，结果发现细胞的融合率、卵裂率以及囊胚率都得到了显著提高。实施例 1、全长线粒体 DNA的扩增 PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) refers to the amplification of a DNA fragment of interest by PCR, and then the fragment to be detected is digested with a restriction endonuclease, and the restriction endonuclease recognizes and cleaves the specific sequence. Then, the digested product is subjected to electrophoresis, and the difference of the gene sequences of different sources is compared according to the size of the DNA fragment. The invention utilizes PCR-RFLP technology to amplify mitochondrial DNA of different cattle, and then performs RPLP analysis to select fragments having different lengths, and divides them into four types according to the restriction enzyme map: A, B, C, and D. Then, the cells with the same haplotype DNA haplotype were selected for nuclear transfer, and the fusion rate, cleavage rate and blastocyst rate of the cells were significantly improved. Example 1. Amplification of full-length mitochondrial DNA

分别抽取不同青年牛外周血标本，肝素抗凝，酚-氯仿分离抽提 DNA, 以抽提的 DNA 为模板，分别以下列 Hl、 H2、 H3、 H4四个片段为引物，扩增不同牛的线粒体 DNA: Peripheral blood samples from different young cattle were extracted, heparin anticoagulation, phenol-chloroform separation and extraction of DNA, and extracted DNA as template, using the following four fragments of Hl, H2, H3 and H4 as primers to amplify different cattle Mitochondrial DNA:

HI : S: 5'-CTGCAGTCTCACCATCAACC-3'； HI : S: 5'-CTGCAGTCTCACCATCAACC-3';

A: 5 '-GTGTAGATGCTTGCATGTAAGT-3 '； A: 5 '-GTGTAGATGCTTGCATGTAAGT-3 ';

H2: S: 5'-TTATCCGTTGGTCTTAGGAA-3'； H2: S: 5'-TTATCCGTTGGTCTTAGGAA-3';

A: 5'GCGGCATGGTAATTAAGCTC-3 '； A: 5'GCGGCATGGTAATTAAGCTC-3 ';

H3: S: 5，-TTATCACAATCCAGAACTGAC-3，； H3: S: 5,-TTATCACAATCCAGAACTGAC-3,;

A: 5，-CTAGTGAGAGTGAGGAGATATG-3，； A: 5,-CTAGTGAGAGTGAGGAGATATG-3,;

H4: S: 5'-TGTGCATGTGACACGTATCC-3'； H4: S: 5'-TGTGCATGTGACACGTATCC-3';

A: 5 '-AAGCGATTGCTTACTAGTCGG-3 '； A: 5 '-AAGCGATTGCTTACTAGTCGG-3 ';

具体扩增条件如下- Specific amplification conditions are as follows -

HI : 94 °C 预变性 5min，进入循环；循环条件为： 94 °C 30s, 58°C30s, 72 °C lmin, 35个循环；最后 72 °C 延伸 10 min。 HI: 94 °C pre-denaturation for 5 min, enter the cycle; cycle conditions are: 94 °C 30s, 58 °C 30s, 72 °C lmin, 35 cycles; the last 72 °C extension for 10 min.

H2: 94 °C 预变性 5min，进入循环；循环条件为： 94 °C lmin, 56°C lmin， 72 °C 4min, 35个循环；最后 72°C 延伸 10 min。 H2: 94 °C pre-denaturation 5 min, enter the cycle; cycle conditions are: 94 °C lmin, 56 °C lmin, 72 °C 4min, 35 cycles; the last 72 °C extension for 10 min.

H3: 94 °C 预变性 5min，进入循环；循环条件为： 94 °C lmin, 56。C lmin， 72 °C 4min， 35个循环；最后 72 °C 延伸 10 min。 H3: 94 °C pre-denaturation for 5 min, enter the cycle; cycle conditions are: 94 °C lmin, 56. C lmin, 72 ° C for 4 min, 35 cycles; last 72 ° C for 10 min.

H4: 94 °C 预变性 5min，进入循环；循环条件为： 94 °C lmin, 56°C lmin， 72 °C 5min， 35个循环；最后 72°C 延伸 10 min。 H4: 94 °C pre-denaturation for 5 min, enter the cycle; The cycle conditions are: 94 °C lmin, 56 °C lmin, 72 °C 5 min, 35 cycles; the last 72 °C extension for 10 min.

扩增完成后均进行 2%琼脂糖凝胶电泳，以检测 PCR产物.，- ^保与预期一致。 After the amplification was completed, 2% agarose gel electrophoresis was carried out to detect the PCR product. The maintenance was consistent with the expectation.

获得的 PCR扩增产物按 PCR产物纯化试剂盒（Takara)产品说明书（产品号 DV807A) 进行纯化。纯化的扩增产物分别以限制性内切酶进行酶切，具体如下: The obtained PCR amplification product was purified by PCR product purification kit (Takara) product specification (product number DV807A). The purified amplification products were digested with restriction endonucleases, respectively, as follows:

HI纯化片段、5μ1 HI纯化片段 5 μΐ NEB buffer4 2 μΐ NEB bufferl 2 μΐ BSA 0.2 μΐ Hpall 0.2 μΐ HI purified fragment, 5μ1 HI purified fragment 5 μΐ NEB buffer4 2 μΐ NEB bufferl 2 μΐ BSA 0.2 μΐ Hpall 0.2 μΐ

MaIII(a、 b) 0.2 μΐ H₂0 12.8 μΐMaIII(a, b) 0.2 μΐ H ₂ 0 12.8 μΐ

H₂0 12.6 μΐ 20 μΐ H ₂ 0 12.6 μΐ 20 μΐ

20 μΐ 20 μΐ

Η2纯化片段 5 μΐ H2纯化片段 5 μΐΗ2 purified fragment 5 μΐ H2 purified fragment 5 μΐ

NEB bufferl 2 μΐ 10XM 2 μΐNEB bufferl 2 μΐ 10XM 2 μΐ

Hpall 0.2 μΐ Pst I 0.2 μΐHpall 0.2 μΐ Pst I 0.2 μΐ

H₂0 12.8 μΐ H₂0 12.8 μΐ H ₂ 0 12.8 μΐ H ₂ 0 12.8 μΐ

20 μΐ 20 μΐ 20 μΐ 20 μΐ

Η3纯化片段 5 μΐ Η3纯化片段 5 μΐ 10XM 2 μΐ 10XK 2 μΐΗ3 purified fragment 5 μΐ Η3 purified fragment 5 μΐ 10XM 2 μΐ 10XK 2 μΐ

Avail 0.2 μΐ BamH I 0.2 μΐAvail 0.2 μΐ BamH I 0.2 μΐ

H₂0 12.8 μΐ H₂0 12.8 μΐ H ₂ 0 12.8 μΐ H ₂ 0 12.8 μΐ

20 μΐ 20 μΐ 20 μΐ 20 μΐ

H4纯化片段 5μ1 H4 purified fragment 5μ1

10XH 2μ1 10XH 2μ1

Bglll 0.2 μΐ Bglll 0.2 μΐ

Η₂0 12.8 μΐ Η ₂ 0 12.8 μΐ

20 μΐ 20 μΐ

其中 Bamin于 30°C酶切过夜，其余均在 37Ό酶切过夜。 Bamin was digested overnight at 30 °C, and the rest were digested overnight at 37 。.

将酶切产物电泳（0.8〜2%琼脂糖， 120V， lh) 以作进一步分析。 The digested product was electrophoresed (0.8~2% agarose, 120V, lh) for further analysis.

实施例 2、酶切片段的分析及线粒体 DNA分类 Example 2. Analysis of restriction fragments and mitochondrial DNA classification

将各种酶 RFLP分析中具有长度差异的片段进行汇总，结果见以下表 1。表 1、内切酶 RFLP分析具有长度差异的片段汇总 Fragments with differences in length in various enzyme RFLP analyses were pooled and the results are shown in Table 1 below. Table 1. Endonuclease RFLP analysis of fragments with length differences

注： +有相应酶酶切位点或具有此酶额外酶切位点; Note: + There is a corresponding enzyme cleavage site or an additional cleavage site with this enzyme;

-无相应酶酶切位点或无此酶额外酶切位点。分析酶切片段的差异，根据不同的个体来源，将不同牛的线粒体单倍型分为 A、 B、 C、 D四种类型，四种类型的酶切图谱如表 2所示： - There is no corresponding enzyme cleavage site or no additional cleavage site for this enzyme. The differences in the fragments were analyzed. According to different individual sources, the mitochondrial haplotypes of different cattle were divided into four types: A, B, C and D. The four types of restriction enzyme maps are shown in Table 2:

表 2、线粒体单倍型酶切图谱 Table 2. Mitochondrial haplotype restriction map

Nlallla Nlalllb Hpall Hpall Pstl Avall BamHl Bglll (HI) (HI) (HI) (H2) (H2) (H3) (H3) (H4) Nlallla Nlalllb Hpall Hpall Pstl Avall BamHl Bglll (HI) (HI) (HI) (H2) (H2) (H3) (H3) (H4)

A - - - -. + + - +A - - - -. + + - +

B - 一 + - + + - +B - one + - + + - +

C + - - + - - + -C + - - + - - + -

D 一 + 一 + + 一 + 实施例 3、受体***的准备 D one + one + + one + Example 3, Preparation of Recipient Oocytes

对单倍型 A型青年牛进行活体取卵，具体方法见申请号为 200510023510.1的中国发明专利申请。 For haplotype A young cattle, the eggs are collected in vivo. For the specific method, see Chinese patent application No. 200510023510.1.

将收集液倒入拣卵杯中，用无钙镁杜氏磷酸缓冲液 (DPBS, 1升去离子水中含 8g NaCl, 0.2g KC1, 1.44g Na₂HP0₄, 0.24g KH₂P0₄, 30g BSA和 2000u肝素）冲洗，拣出***复合体（cumulus oocyte complexes, COCs)移入培养皿，在体视显微镜下对 COCs进行分级，选择胞质均匀，周围卵丘细胞排列紧密，包绕三层以上的***，放入成熟液（TCM-199 (Gibco, Grand Island, NY)外加 10%胎牛血清（FBS )、 l(^g/ml促黄侔生成激素、 l g/ml ***和 ^g/ml促卵泡生成素）中培养 15〜30h。实施例 4、供核体细胞的准备 Pour the collected liquid into the picking cup, using calcium-free magnesium Duchentate buffer (DPBS, 1 liter of deionized water containing 8g NaCl, 0.2g KC1, 1.44g Na ₂ HP0 ₄ , 0.24g KH ₂ P0 ₄ , 30g BSA Rinse with 2000u heparin, remove the cumulus oocyte complexes (COCs) into the culture dish, classify the COCs under a stereo microscope, select the cytoplasm to be uniform, and arrange the surrounding cumulus cells closely, covering the three layers. The above oocytes were placed in mature solution (TCM-199 (Gibco, Grand Island, NY) plus 10% fetal bovine serum (FBS), l (^g/ml jaundice-producing hormone, lg/ml estradiol and Incubate for 15 to 30 h in ^g/ml follicle stimulating hormone). Example 4 Preparation for nuclear cells

分别取经以上线粒体 DNA单倍型分类的青年牛成纤维细胞或卵丘细胞，用含 10% FBS的 DMEM/F12 (Gibco公司，产品号分别为 11039-021 ) 进行培养。 Young bovine fibroblasts or cumulus cells classified by the above mitochondrial DNA haplotype were cultured in DMEM/F12 (Gibco, product number 11039-021, respectively) containing 10% FBS.

1、细胞原代培养 1. Primary culture of cells

①成纤维细胞的原代培养 Primary culture of fibroblasts

取新生牛耳组织，置于含双抗（2%青链霉素）的 0.9%生理盐水中，耳组织经碘酊和 75%酒精浸泡消毒后，无菌生理盐水洗 3〜5遍，剪碎，加入 0.25 %胰蛋白酶， 37°C消化 2 h，加入含 10%FBS的 DMEM/F12培养基， 37°C、 5 %C0₂、饱和湿度培养箱中原代培养，倒置显微镜下观察细胞贴壁后更换培养基继续培养。 Take the newborn ear tissue, place it in 0.9% normal saline containing double antibody (2% streptomycin), sterilize the ear tissue with iodine and 75% alcohol, wash it 3 to 5 times in sterile saline, and cut it. Add 0.25% trypsin, digest at 37 °C for 2 h, add DMEM/F12 medium containing 10% FBS, primary culture in 37 ° C, 5 % CO ₂ , saturated humidity incubator, observe the cell adherence after inverted microscope Change the medium and continue to culture.

② 卵丘细胞的原代培养 2 Primary culture of cumulus cells

将成熟 20h小时的 COCs (见实施例 3 )放入含 0.5%透明质酸酶的无钙镁的 DPBS中，消化 l〜2min，同时用吸胚管反复吹吸，脱去外层疏松、扩展的卵丘细胞。 The mature 20h hour COCs (see Example 3) were placed in calcium-free magnesium-containing DPBS containing 0.5% hyaluronidase, digested for 1~2 min, and repeatedly sucked with a suction tube to remove the outer layer loose and expand. Cumulus cells.

收集这部分的卵丘细胞，尽快移入无钙镁的 DPBS中， 1200rpm离心 5min，弃上清；加入 ΙΟΟμΙ 0.25 %胰酶吹打 lmin，加入 DMEM/F12 + 10 %FBS ( 1 体积 FBS+9 体积 DMEM/F12培养液）终止消化， lOOOrpm离心 5min， DMEM/F12培养液洗涤 2次。用 DMEM/F12+ 10%FBS悬浮细胞，轻轻吹打，使细胞充分吹散，接种至 25cm²培养瓶（接种密度约为 I X 10⁵，内含 DMEM/F12+ 10%FBS培养液 5ml)，于 38.5°C， 5%C0₂, 饱和湿度的培养箱中培养。 Collect this part of the cumulus cells, transfer to calcium-free magnesium DPBS as soon as possible, centrifuge at 1200 rpm for 5 min, discard the supernatant; add ΙΟΟμΙ 0.25 % trypsin for 1 min, add DMEM/F12 + 10 % FBS (1 volume FBS + 9 volume DMEM) /F12 medium) The digestion was terminated, centrifuged at 100 rpm for 5 min, and washed twice with DMEM/F12 medium. The cells were suspended in DMEM/F12+ 10% FBS, gently pipetted, and the cells were thoroughly blown off, and inoculated into a 25 cm ² flask (inoculation density of about IX 10 ⁵ , containing DMEM/F12 + 10% FBS medium 5 ml), at 38.5 Incubate in an incubator at °C, 5% C0 ₂ , saturated humidity.

2、约 3〜5天生长汇合后，进行传代。 2. After about 3 to 5 days of growth and convergence, pass through.

3、取培养 1〜5代的细胞用作核移植的供核细胞。细胞生长至瓶底 80〜90%时，将培养液换成含 0.5%FBS的培养液血清饥饿 2〜3天，诱导细胞进入 G0/G1期。 3. Cells cultured for 1 to 5 passages are used as donor cells for nuclear transfer. When the cells were grown to the bottom of the bottle at 80 to 90%, the culture solution was replaced with a culture solution containing 0.5% FBS and starved for 2 to 3 days to induce the cells to enter the G0/G1 phase.

核移植前，用 0.25 %胰酶消化贴壁细胞，将消化下来的细胞用 3mg/ml的链霉蛋白酶处理 50秒，并离心洗涤，最后用 DMEM/F12培养液（GIBICO公司） ΙΟΟμΙ重悬，并反复吹打成单个细胞的悬液，保存于恒温箱中待用。实施例 5、细胞核移植 Prior to nuclear transfer, adherent cells were digested with 0.25% trypsin, and the digested cells were treated with 3 mg/ml of pronase for 50 seconds, washed by centrifugation, and finally resuspended in DMEM/F12 medium (GIBICO) ΙΟΟμΙ. The suspension of individual cells is repeatedly blown and stored in an incubator for use. Example 5, nuclear transfer

5.1 、核移植所需 ACM培养液的配制 5.1, preparation of ACM culture solution for nuclear transfer

ACM培养液的配方如下: The formula of ACM culture solution is as follows:

NaCl (Sigma S-5886) 0.580 g NaCl (Sigma S-5886) 0.580 g

KC1 ( Sigma P-5405 ) 0.022 g KC1 ( Sigma P-5405 ) 0.022 g

NaHC0₃ (Sigma S-5761 ) 0.209 g NaHC0 ₃ (Sigma S-5761 ) 0.209 g

L-谷氨酰胺（ Sigma G-8540) 0.015 g L-Glutamine (Sigma G-8540) 0.015 g

CaCl₂ · 2H₂0 ( Sigma C-7902 ) 0.004 g CaCl ₂ · 2H ₂ 0 ( Sigma C-7902 ) 0.004 g

丙酮酸（2.2 mg/ml贮存于 PBS中） 2ml Pyruvic acid (2.2 mg/ml in PBS) 2ml

BME氨基酸（ Sigma B-6766) 2ml BME Amino Acid ( Sigma B-6766) 2ml

MEM氨基酸（ Sigma M-7145) 1ml MEM Amino Acid ( Sigma M-7145) 1ml

青链霉素（GIBICO-BRL公司） 1ml Streptomycin (GIBICO-BRL) 1ml

乳酸（ Sigma L-7900) 14μ1 Lactic acid (Sigma L-7900) 14μ1

酚磺酞（ Sigma P-0290) ΙΟΟμΙ Phenolsulfonate (Sigma P-0290) ΙΟΟμΙ

BSA (无脂肪酸）（ Sigma A-6003 ) 0.300 g 将除 BSA外的所有干粉成分溶解于盛有 70ml胚胎水的烧杯中，为防止 BME/MEM 受到污染，加入所有液体成分并用罩子罩住烧杯进行搅拌，然后加入 BSA干粉继续搅拌直至完全溶解，用量筒定容至 100ml，最后用 0.2μπι的滤膜过滤，于 4Ό贮存不超过两周。 BSA (no fatty acid) ( Sigma A-6003 ) 0.300 g Dissolve all dry powder ingredients except BSA in a beaker containing 70 ml of embryonic water. To prevent contamination of BME/MEM, add all liquid ingredients and cover the beaker with a cover. Stir, then add BSA dry powder and continue to stir until completely dissolved. The volume is adjusted to 100 ml, finally filtered through a 0.2 μm filter and stored at 4 Torr for no more than two weeks.

5.2、细胞核移植 5.2, nuclear transfer

用内径约 20微米的去核针将受体***的第一极体及其附近区域的卵核去除，然后将不同线粒体 DNA 单倍型的供体细胞核注入去核***卵周隙，以 2.5Ky/cm— 3V/cm和 6— 10μ3的融合参数，将供核细胞核融入***，获得克隆重构胚。融合操作后的卵移入 ACM培养液中，于 5 %C0₂、 38.5 °C ,饱和湿度的培养箱中培养。 30〜60min后，在体视显微镜下检査融合情况，计算融合率，结果如表 3所示。 The nucleus of the first polar body of the recipient oocyte and its vicinity is removed by a denucleation needle with an inner diameter of about 20 μm, and then the donor nucleus of different mitochondrial DNA haplotypes is injected into the perivitelline space of the enucleated oocyte. The nuclear nucleus was integrated into the oocyte at a fusion parameter of 2.5 Ky/cm - 3 V/cm and 6 - 10 μ3 to obtain a cloned reconstructed embryo. The eggs after the fusion operation were transferred to an ACM culture solution, and cultured in an incubator at 5 % C0 ₂ , 38.5 ° C, and a saturated humidity. After 30 to 60 min, the fusion was examined under a stereo microscope, and the fusion rate was calculated. The results are shown in Table 3.

随后，用离子霉素（Ionomycin, Sigma 1-0634) 5ng/ml激活上述融合的卵，然后放入含 CHX+CB (两者都购自 sigma公司）的培养盘，并在 38.5°C、饱和湿度的培养箱中放置 5小时。 Subsequently, the above fused eggs were activated with ionomycin (Ionomycin, Sigma 1-0634) 5 ng/ml, and then placed in a culture dish containing CHX+CB (both purchased from sigma) and saturated at 38.5 °C. Place in a humidified incubator for 5 hours.

重构胚放入 ACM培养液和胎鼠成纤维细胞（MEF) +1 %胎牛血清（FBS) 进行共培养，在 5 %C0₂、 38.5°C，饱和湿度的培养箱中培养。 72h后更换培养液和 MEF细胞 +10 %FBS。 48h观察重构胚的卵裂情况，计算卵裂率，培养第 7天记录囊胚的数量，计算囊胚率，结果如表 3所示。表 3、核质间不同单倍型组合与核移植效率的比较 The reconstructed embryos were cultured in ACM medium and fetal rat fibroblasts (MEF) +1% fetal bovine serum (FBS), and cultured in an incubator at 5 % C0 ₂ , 38.5 ° C, and saturated humidity. After 72 h, the culture medium and MEF cells + 10% FBS were replaced. The cleavage condition of the reconstructed embryos was observed at 48h, the cleavage rate was calculated, and the number of blastocysts was recorded on the 7th day of culture, and the blastocyst rate was calculated. The results are shown in Table 3. Table 3. Comparison of different haplotype combinations and nuclear transfer efficiency between nucleoplasms

注：融合率 =融合数 /重构胚数 Note: Fusion rate = number of fusions / number of reconstructed embryos

卵裂率 =培养 48小时***数 /培养数 Cleavage rate = culture 48 hours split number / culture number

囊胚率 =培养 7天所得囊胚数 /卵裂数根据表 3的结果，分别统计相同线粒体 DNA单倍型（同型）之间的核移植效率以及不同线粒体 DNA单倍型之间（异型）的核移植效率，结果如表 4所示。表 4、不同类型核移植效率的比较 Blastocyst rate = number of blastocysts/cleavage number obtained in 7 days of culture According to the results in Table 3, the nuclear transfer efficiency between the same mitochondrial DNA haplotype (homotype) and the difference between different mitochondrial DNA haplotypes (heterotype) were counted separately. The nuclear transfer efficiency, the results are shown in Table 4. Table 4. Comparison of different types of nuclear transfer efficiency

δ 从以上表 3的数据可以看出，不同组合方式的融合率和卵裂率基本一致，均在 70% 和 54%上下；而囊胚率方面，相同单倍型之间的组合（A— A和 C—C组合）效果最佳，均达到了 40%以上，明显优于不同单倍型之间的组合（为 30%左右）。其中 A— A组合的囊胚发育率最髙，达到 49.8 % ; C— C组合次之，达到 42.2% ; 然后是 A^C组合，达到 36.1 % , C— A组合发育率最低，为 24.4%。

δ As can be seen from the data in Table 3 above, the fusion rate and cleavage rate of different combinations are basically the same, both above 70% and 54%; and in terms of blastocyst rate, the combination between the same haplotypes (A-A The combination with C-C) has the best effect, all of which are above 40%, which is obviously better than the combination between different haplotypes (about 30%). The blastocyst development rate of A-A combination was the highest, reaching 49.8 %; the C-C combination was second, reaching 42.2%; then the A^C combination was 36.1%, and the C-A combination development rate was the lowest, 24.4%. .

从表 4综合比较的结果也可以非常清楚地看到，与异型核移植相比，两者间的融合率和卵裂率相差不大，但是同型核移植的囊胚率明显高于异型核移植，约为异型核移植的 1.5倍，说明同型核植相比异型核移植更易跨过胚胎体外发育的阻滞期，从而表现出发育优势。 From the results of the comprehensive comparison in Table 4, it can be clearly seen that the fusion rate and cleavage rate are not much different from those of heterotypic nuclear transfer, but the blastocyst rate of homonuclear transplantation is significantly higher than that of heterotypic nuclear transfer. , about 1.5 times of heterotypic nuclear transfer, indicating that isotype nuclear transfer is easier to cross the in vitro development of the embryo than the heterogeneous nuclear transfer, thus showing a developmental advantage.

综上所述，采用线粒体 DNA单倍型相同的细胞核和***细胞质进行核移植，可以有效提高体细胞核移植的效率。采用本发明的方法可以克服现有技术中由于***来源不清所导致的细胞核移植效率低下的问题。虽然以上仅以牛为例对本发明的方法进行了验证，但是本领域的技术人员根据说明书的内容，显然可以看出本发明的方法同样适用于其它非人哺乳动物，例如猪、羊以及老鼠等，因此，针对这些非人哺乳动物的同型核移植方法同样应当属于本发明的范围。 In summary, nuclear transfer using the same nucleus and cytoplasm of mitochondrial DNA haplotypes can effectively improve the efficiency of somatic cell nuclear transfer. The use of the method of the present invention overcomes the problem of inefficient prior art nuclear transfer due to unclear oocyte origin. Although the method of the present invention has been verified by taking the cattle as an example, those skilled in the art can clearly see that the method of the present invention is equally applicable to other non-human mammals, such as pigs, sheep, and mice, according to the contents of the specification. Therefore, a homologous nuclear transfer method for these non-human mammals should also fall within the scope of the present invention.

Claims

权利要求书 Claim

1、一种细胞核移植方法，其特征在于，该方法是以线粒体单倍型相同的非人哺乳动物的供核体细胞和受体***进行核移植。 A method for nuclear transfer, characterized in that the method is a nuclear transfer of a donor cell and a recipient oocyte of a non-human mammal having the same mitochondrial haplotype.

2、如权利要求 1所述的方法，其特征在于，包括以下步骤- a、选取某一线粒体 DNA单倍型的非人哺乳动物，以其成皮肤成纤维细胞或卵丘细胞作为供核体细胞； 2. The method according to claim 1, comprising the steps of: a, selecting a non-human mammal of a mitochondrial DNA haplotype, and using the dermal fibroblast or cumulus cell as a donor nucleus Cell

b、选取相同线粒体 DNA单倍型的同一种哺乳动物，以其***作为受体细胞； c、受体***去核； b. The same mammal of the same mitochondrial DNA haplotype is selected, and the oocyte is used as a recipient cell; c. The recipient oocyte is enucleated;

3、如权利要求 2所述的方法，其特征在于，作为供核体细胞的成纤维细胞或卵丘细胞是经过加 10%胎牛血清的 DMEM/F12培养 1一 5代，并 0.5%胎牛血清饥饿 2— 3天的细胞。 3. The method according to claim 2, wherein the fibroblast or cumulus cells serving as the nuclear somatic cells are cultured for 1 to 5 passages in DMEM/F12 supplemented with 10% fetal bovine serum, and 0.5% of the fetuses. Bovine serum starved for 2-3 days of cells.

4、如权利要求 2所述的方法，其特征在于，作为受体细胞的***通过活体取卵的方法获得。 4. The method according to claim 2, wherein the oocyte as the recipient cell is obtained by a method of taking an egg in vivo.

5、如权利要求 2所述的方法，其特征在于，所述***去核是将***的第一极体及其附近区域的卵核去除。 ' 5. The method according to claim 2, wherein the oocyte enucleation is to remove the egg nucleus of the first polar body of the oocyte and its vicinity. '

6、如权利要求 2所述的方法，其特征在于，所述细胞核融入***的融合参数为 2.5KV/cm-3V/cm, 6—10μβ。 6. The method according to claim 2, wherein the fusion parameter of the nuclear nucleus into the oocyte is 2.5 KV/cm-3 V/cm, 6-10 μβ.

7、如权利要求 1所述的方法，其特征在于，所述非人哺乳动物包括：牛、羊、猪和老鼠。 7. The method of claim 1, wherein the non-human mammal comprises: cattle, sheep, pigs, and mice.

8、如权利要求 7所述的方法，其特征在于，所述哺乳动物为牛。 8. The method of claim 7 wherein the mammal is a cow.

9、如权利要求 1一 8中任一项所述的方法，其特征在于，还包括对哺乳动物的线粒体 DNA进行单倍型分析的步骤。 The method according to any one of claims 1 to 8, further comprising the step of performing haplotype analysis on mammalian mitochondrial DNA.

10、如权利要求 9所述的方法，其特征在于，所述单倍型分析是通过 PCR— R LP技术。 10. The method of claim 9, wherein the haplotype analysis is by a PCR-R LP technique.