WO2013159313A1 - 一种动物胚胎干细胞系及其制备方法和应用 - Google Patents
一种动物胚胎干细胞系及其制备方法和应用 Download PDFInfo
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- the invention relates to a stem cell and a preparation method and application thereof, in particular to an animal embryonic stem cell line, a preparation method and application thereof, and belongs to the technical field of cell biology and developmental biology. Background technique
- Transgenic animals provide an important research model for modern medical and biological research, but it is still very difficult to obtain transgenic animals from mammals.
- transgenic animals are obtained mainly by pronuclear microinjection or by transgenic animals using embryonic stem cells and homologous recombination techniques.
- the pronuclear microinjection method is to introduce the exogenous target fragment into the male pronucleus. This method has always been considered as a classic scheme for the development of transgenic animals, but it has great limitations, which are highlighted by the exogenous gene integration site.
- the object of the present invention is to provide an animal embryonic stem cell line and a preparation method and application thereof, which can quickly, directly and efficiently obtain a transgene, in view of the inefficiency of the current transgenic animal production technology and the inability to widely spread and apply it. animal.
- the present invention also opens up a rapid and straightforward method for producing transgenic animals, making the production of genetically modified animals of the disease model simpler.
- the invention provides a mouse haploid embryonic stem cell line
- the cell line is a solitary male haploid embryonic stem cell line N-1-1 deposited under the accession number CGMCC No. 6037 (the classification of the cell line) Named as mouse haploid embryonic stem cells, and deposited with the China Microbial Culture Collection Management Committee General Microbiology Center (CGMCC) on April 19, 2012, Address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China Institute of Microbiology, Academy of Sciences; Zip Code: 100101).
- CGMCC China Microbial Culture Collection Management Committee General Microbiology Center
- the mouse haploid embryonic stem cell line can differentiate into cells of three germ layers, and more preferably, the cells of the three germ layers are ventricles, muscles and glands.
- the present invention provides a rat haploid embryonic stem cell line, which is a solitary male haploid embryonic stem cell line HD2-2 deposited under the accession number CGMCC No. 6038 (the cell line is classified as Rat haploid embryonic stem cells were deposited with the General Microbiology Center (CGMCC) of the China Microbial Culture Collection Committee on April 19, 2012. Address: No. 3, No.1, Beichen West Road, Chaoyang District, Beijing, China Institute; Zip Code: 100101).
- CGMCC General Microbiology Center
- the rat haploid embryonic stem cell line can differentiate into cells of three germ layers, and more preferably, the cells of the three germ layers are ventricles, muscles and glands.
- the present invention provides an animal transgenic embryonic stem cell line, which is a plasmid of a GFP gene linked by an EF1 a promoter, which is transfected into a mouse haploid embryonic stem cell line or the present invention
- the rat haploid embryonic stem cell line of the invention is obtained.
- the present invention provides a method of preparing an animal embryonic stem cell line, comprising the steps of:
- the mulberry or embryos cultured in the step 2) are first inoculated on the feeder cells, and then inoculated into the embryonic stem cell culture solution to prepare primary solitary male haploid embryonic stem cells;
- step 4) until a lone male haploid animal embryonic stem cell line with a haploid ratio of 85% to 100% is obtained.
- the sperm cell is a tailed sperm head, and the tailed sperm head is injected into the oocyte in the M2 operating solution; preferably, the animal sperm cell and the animal egg
- the mother cell is derived from mouse or rat.
- the female nucleus is removed by treatment with HCG hormone super-discharge for 20 hours.
- step 2) the orphan male haploid embryo is activated in KSOM-AA culture medium containing 5 ⁇ g/ml cytochalasin for 5-6 hours, and then the activated solitary male haploid embryo is activated.
- the cells were transferred to KSOM-AA broth without cytochalasin for 72 hours in vitro to form mulberry abortion.
- step 2) the solitary male haploid embryo is transplanted into a 0.5 dpc pseudopregnant animal oviduct, and the embryo is formed 96 h later.
- the feeder layer cells are prepared from embryonic fibroblasts, and the embryonic stem cell culture solution comprises N2B27 culture solution and an additive, preferably, when sperm cells and oocytes are derived from mice
- the additive comprises Knockout serum replacement (KOSR), leukemia inhibitory factor, MEK inhibitor, GSK3 beta inhibitor, and inhibitor of cyclin 53; or
- the additives include ⁇ 27632, A83-01, CHIR99021 and PD0325901;
- the additive further comprises a growth factor, and most preferably, the growth factor is a rat growth factor or a mouse growth factor.
- the present invention provides a method for preparing a transgenic animal, comprising the steps of: transfecting a plasmid of a GFP gene linked to an EF1 ⁇ promoter into an animal embryo prepared by the method for preparing an animal embryonic stem cell line of the present invention;
- the stem cell line is used to prepare an animal transgenic embryonic stem cell line, and the animal transgenic embryonic stem cell line is injected into the cytoplasm of the oocyte to obtain a reconstructed embryo, and then the reconstructed embryo is transplanted into the pseudopregnant animal, and after the pseudopregnancy development,
- the transgenic animal is obtained, preferably, the reconstructed embryo is transplanted into a fallopian tube of a 0.5 dpc pseudopregnant animal; preferably, the pseudopregnant animal is a mouse or a rat.
- the animal transgenic embryonic stem cell line is injected into the cytoplasm of the pre-activated sputum oocyte by micromanipulation to obtain a reconstructed embryo, and then the reconstructed embryo is activated and cultured to a diploid in vitro;
- the oocytes of the flood stage are preactivated by SrCl 2 for 20 minutes;
- the pseudopregnant animal is a mouse
- the reconstructed embryo is activated with a CZB activating solution containing SrCl 2 for 3 hours; or
- the reconstructed embryo is activated with 150 ⁇ M of Butyrolactone I (BLI) activating solution (purchased from BIOMOL) for 3 hours;
- BLI Butyrolactone I
- the reconstructed embryo is cultured in vitro for 24 hours to diploid.
- the animal transgenic embryonic stem cell line is further included before being injected into the cytoplasm of the oocyte.
- the G0/G1 phase cell line is obtained by in vivo staining the animal transgenic embryonic stem cell line and then screened by flow cytometry; the nuclear metaphase cell line is pretreated with colchicine by transgenic embryonic stem cell line 3 It is obtained by pretreatment for 10 hours or by using nocodazole.
- the present invention provides a preparation method of the mouse haploid embryonic stem cell line of the present invention or the rat haploid embryonic stem cell line of the present invention or the animal embryonic stem cell line of the present invention.
- the use of the animal embryonic stem cell line in the preparation of a transgenic animal kit preferably, the transgenic animal kit is a transgenic mouse kit or a transgenic animal kit.
- the present invention provides an animal prepared by the mouse haploid embryonic stem cell line of the present invention or the rat haploid embryonic stem cell line of the invention or the preparation method of the animal embryonic stem cell line of the invention.
- the present invention provides a transgenic animal kit or a cell differentiation kit, comprising the mouse haploid embryonic stem cell line of the present invention or the rat haploid embryonic stem cell line of the present invention or the present invention
- An animal embryonic stem cell line prepared by the method for preparing an animal embryonic stem cell line.
- the orphan male haploid animal embryonic stem cell line of the present invention not only has the basic characteristics of embryonic stem cells, but also enables the oocytes to be fertilized and then developed into a complete animal individual. Therefore, from the perspective of producing transgenic animals, ahESCs can be genetically targeted, and then the offspring of the animal with specific genetic modification can be obtained by cytoplasmic injection of oocytes.
- the invention relates to a method for developing a transgenic animal by using a lone male haploid embryonic stem cell line.
- the ahESCs can be efficiently targeted and targeted, and the transgenic haploid stem cells can be amplified by using the characteristics of rapid self-renewal of stem cells;
- the ability of oocytes to fertilize in vitro into animal individuals inherit the genetic characteristics of genetic modification to the next generation, and obtain the transgenic animals quickly, directly and efficiently by crossing the self-crossing of the first generation.
- Embryonic stem cell lineage is inherited, which greatly shortens the cycle of obtaining pure-transgenic animals.
- the present invention establishes a base of mouse and rat rodent ahESCs, enabling them to inherit to the next generation, which not only lays a foundation for the development of innovative assisted reproductive technology, but also opens up a rapid production of transgenic animals.
- the direct method makes the production of transgenic animals in disease models more simple and convenient.
- the new method from transgenic haploid cells directly to transgenic animals can not only help the eugenics of large animals, but also breed high-quality new varieties. At the same time, it can improve the genetic diseases of the fathers from the perspective of cells, and even accelerate the evolution of species.
- Figure 1 shows the results of a haploid sorting by a flow cytometry of a haploid embryonic stem cell line according to the present invention.
- P2 represents a G0/G1 phase haploid embryonic stem cell line; Mouse haploid embryonic cells;
- Figure 3 shows the mouse haploid embryonic stem cell line of the present invention
- Figure 4 shows the karyotype of the orphan male haploid embryonic stem cell line of the present invention
- Figure 5 shows the results of immunofluorescence staining of molecular markers of different pluripotency of the orphan male haploid embryonic stem cells of the present invention.
- Fig. 5A is a test result of expressing Oct3/4 by immunofluorescence staining
- Fig. 5B is a test result of expressing Nanog by immunofluorescence staining
- Fig. 5C is a test result of expressing Sox2 by immunofluorescence staining
- Fig. 5D is an expression of SSEA- expressing SSEA- 1 test results by immunofluorescence staining;
- Figure 6 shows the orphan male haploid embryonic stem cell line of the present invention having the differentiation potential of the three germ layers, wherein Figure 6A is the ventricle formed by the differentiation of the orphan male haploid embryonic stem cell line of the present invention; The muscle formed by differentiation of the orphan male haploid embryonic stem cell line of the invention; FIG. 6C is a gland formed by differentiation of the orphan male haploid embryonic stem cell line of the present invention;
- Figure 7 shows the haploid progeny obtained after the orphan male haploid embryonic stem cell line of the present invention is injected into the oocyte plasma cells;
- Figure 8 shows the results of SSLP genetic analysis of the haploid progeny mouse fetus of the present invention, wherein Figure 8A shows the electrophoresis result of D7Mit44 in a PCR-amplified mouse sample; Figure 8B shows the D8Mit94 in a PCR-amplified mouse sample. Electrophoresis results; Figure 1 shows the DNA marker, 2 indicates the PCR product of the CD-I mouse tail cell sample, 3 indicates the PCR product of the C57BL/6 mouse tail cell sample, and 4 is the DB A/2 mouse tail.
- PCR product of a sharp cell sample 5 is a PCR product of a 129Sv/Jae mouse tail cell sample, and 6 is a PCR product of a lone male haploid embryonic stem cell line (N-1 - 1 ) sample, 7 a PCR product of a fetal tail tip cell sample of a haploid progeny mouse;
- Figure 9 shows a map of the modified Plenti6.0 linearized vector
- Figure 10 shows a green fluorescent protein color map of the mouse transgenic embryonic stem cell line of the present invention
- Fig. 11 shows the results of a haploid sorting by a flow cytometry of the mouse transgenic embryonic stem cell line of the present invention.
- P2 indicates a G0/G1 phase and a midnuclear haploid embryonic stem cell line.
- the mouse embryonic stem cell with the accession number is CGMCC No. 6037.
- the cell line is classified as mouse haploid embryonic stem cells and deposited on April 19, 2012 at the General Microbiology Center of China Microbial Culture Collection Management Committee (CGMCC). ), Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, China; Zip code: 100101.
- the rat embryonic stem cell with the accession number is CGMCC No. 6038.
- the cell line is classified as rat haploid embryonic stem cell and deposited with the General Microbiology Center of China Microbial Culture Collection Management Committee (CGMCC) on April 19, 2012. ), Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, China; Zip code: 100101.
- CGMCC General Microbiology Center of China Microbial Culture Collection Management Committee
- test materials used in the following examples were mice of the CD-I ( ICR ), C57BL/6, DBA/2, 129Sv/Jae and SCID strains, and the DA and SD strains were purchased from Beijing Vitallihua experimental animals. Technology Co., Ltd.
- mice of the B6D2F1 strain were mated from purchased C57BL/6 and DBA/2 strain mice, and mouse embryonic fibroblasts were purchased from the Institute of Zoology, Chinese Academy of Sciences.
- the formulas of M2 operating solution (purchased from Sigma), KSOM-AA medium and CZB activator are as follows: Table 1 Components and content required to prepare 100 ml KSOM (culture solution)
- Mouse haploid embryos Lonely male haploid embryo remodeling was performed by Qi Zh0U's "one-step" nuclear transfer technique (1) . Specifically, 8 weeks old 129Sv/Jae male rats were sacrificed by cervical dislocation, and sperm in the vas deferens were taken. The sperm is interrupted in the tail of the ultrasonic oscillator, leaving the sperm head, which serves as donor cells for nuclear transfer. Nuclear-transplanted oocytes were obtained from 8-week-old B6D2F1 female rats that had undergone super-discharge treatment.
- the oscillating operation is carried out in the M2 operating solution, the pretreated sperm head is injected into the oocyte, and the spindle of the oocyte is aspirated, and the reconstructed embryo is reconstituted. It is called Androgenetic haploid embryo (AHE).
- AHE Androgenetic haploid embryo
- the reconstructed haploid embryos are activated in KSOM-AA medium containing 5 g/ml cytochalasin (CB) (the sperm itself activates the oocytes), and the purpose of CB is to prevent polar bodies.
- CB cytochalasin
- the haploid embryos were transferred to CB-free KSOM-AA culture medium for in vitro culture at 37 ° C, 5% C0 2 .
- part of the embryos develop to the morula, and these morulas are implanted into the embryonic stem cell culture medium to establish a haploid embryonic stem cell line.
- Rat haploid embryo a method of obtaining a fertilized egg to the female pronucleus by a solitary male haploid remodeling. Specifically, the fertilized eggs of the DA and SD lines were inoculated in the body, and the denuclearization operation was carried out 20 hours after the HCG hormone super-discharge treatment. The reconstituted solitary male embryos were transplanted into the fallopian tubes of 0.5 dpc pseudopregnant mothers, and 96 hours later, the rat solitary haploid embryos were washed.
- Example 2 Establishment of a lone male haploid embryonic stem cell line (ahESCs)
- the reconstituted AHE mulberry embryo was used to efficiently establish the ahESC cell line.
- haploid embryos were inoculated on feeder cells, which were prepared using mouse embryonic fibroblasts.
- the culture medium for establishing the haploid embryonic stem cell line is composed of N2B27 basic culture solution (GIBCO) and various additives.
- the N2B27 basic culture solution is reported by Ying QL et al. (2) , and the additive includes Knockout serum replacement (KOSR).
- GBCO N2B27 basic culture solution
- the clones were picked about 6 days after the embryo was inoculated, and the clones were digested with 0.25% trypsin. After digestion, the culture was continued to establish a haploid stem cell line. At this time, the cells were recorded as primary solitary haploid embryonic stem cells (P1). ). Haploid embryonic stem cells were passaged once every 2 to 3 days. When haploid cells are passed to passages 4 to 5, haploid cells in the cell line are purified by flow cytometry.
- the method of flow screening mainly refers to the haploid purification office of Elling U et al.
- Method (3) the actual 3 whole sputum was treated with ⁇ gml" 1 Hoechst33342 and 50 ⁇ Verapamil (Sigma) for 30 minutes, then subjected to haploid cell sorting (3) .
- the haploid cell line was subjected to haploid purification to obtain a long-term stable solitary haploid embryonic stem cell line, which is a mouse haploid embryonic stem cell line.
- the results are shown in Figure 1.
- the haploid ratio was 85% or more.
- Haploid embryonic stem cell line The mouse haploid embryonic stem cell line (ie, mouse haploid embryonic stem cell line with accession number CGMCC No. 6037) was observed by a living cell workstation (Leica).
- the mouse haploid embryonic stem cell line has a clone morphology similar to that of diploid mouse stem cells (ESCs).
- the rat haploid embryonic stem cell line has the same establishment system as the mouse, except that the mouse growth factor is replaced with rat growth factor based on the medium of the mouse, and the additive is changed to Y27632 (MERCK , A83-01 (Stemgent), CHIR99021 (Stemgent) and PD0325901 (Stemgent) four growth factors, the rat haploid embryonic stem cell line, the rat haploid embryonic stem cell line with the accession number CGMCC No.6038 , the specific results are not shown.
- Example 3 Stem cell function of 3 ⁇ 4 haploid stem cell line ⁇
- Example 2 The solitary male haploid embryonic stem cell line obtained in Example 2, which is a mouse haploid embryonic stem cell line, was detected by the method of G-band karyotype (3) . The result is shown in Fig. 4, and its karyotype is 19+. X.
- the purified solitary male haploid embryonic stem cell line still expresses pluripotency molecular markers, such as Oct3/4, Nanog, Sox2, SSEA-1, etc. (6) , first fixes the cells with 4% polyfurfural. Minutes, then add 0.5% Triton X-100 for 30 minutes, add primary antibody: Anti- Oct3/4
- the orphan male embryonic stem cell line obtained in Example 2 was injected into the oocyte cytoplasm through the intracytoplasmic injection (ICSI) test and the round sperm intracytoplasmic injection (ROSI) experiment (4) .
- ICSI intracytoplasmic injection
- ROSI round sperm intracytoplasmic injection
- Haploid donor cells are divided into two types: G0/G1 phase cells and nuclear metaphase cells. G0/G1 phase cells are stained by Hoechst33342 in vivo and accurately screened by flow cytometry, while nuclear metaphase cells pass through colchicum. Pretreatment for 3 hours or pretreatment with nocodazole for 10 hours. The two types of donor cells were injected into the oocyte cytoplasm of the CD-I mice of the sputum stage of S. cerevisiae for 20 minutes by microinjection into SrCl 2 and activated by SZCl 2 -containing CZB activator for 3 hours.
- the activated reconstructed embryos were transferred into KSOM-AA culture medium and cultured in vitro at 37 ° C, 5% CO 2 . After 24 hours of in vitro culture, the cleavage rate was counted and all 2 times somatic embryos were transplanted into 0.5 dpc (0.5 day) pseudopregnant mice.
- the pseudopregnant mice were delivered or caesarean section after 19.5 days of development, and the haploid progeny mouse fetuses were obtained. The results are shown in Fig. 7.
- the intracytoplasmic injection of oocytes in rats was similar to that of mice, but the activation solution was changed to a lactone I activator (butyrolactonel activator) with a final concentration of 150 ⁇ , which was 21.5 days in pregnancy, and healthy offspring were obtained.
- lactone I activator butyrolactonel activator
- SSLP genetic analysis was performed by microsatellite marker molecules D7Mit44, D8Mit94( 5 ) to identify the solitary haploid embryonic stem cell line and its offspring from ICAI.
- mice of CD-I ICR
- C57BL/6 C57BL/6
- DBA/2 129Sv/Jae strains
- haploid mouse haploid embryonic stem cell line prepared in Example 2 was obtained in Example 4.
- the tip of the haploid mouse fetus, extract DNA, perform PCR amplification, and the primers are from
- the amplification conditions were 94 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 30 seconds, amplification for 35 cycles, PCR amplification, and electrophoresis of the PCR product, color analysis, Figure 8A for amplification
- the test results of D7Mit44 in the sample, Figure 8B is the test result of D8Mit94 in the amplified sample, indicating that the offspring mouse has both the solitary male haploid embryonic stem cell line (129Sv/Jae) and the oocyte (CD-I (ICR)).
- Two sets of genomes indicate that the solitary male haploid embryonic stem cell line ICAI progeny mice were indeed derived from the corresponding haploid embryonic stem cells.
- Example 6 Transgenic modification of haploid embryonic stem cell lines
- the GD fluorescent protein was used as an example to transgenic the solitary male haploid embryonic stem cell line.
- the CMV promoter carried by the Plenti6.0 vector purchased from Invitrogen
- the engineered plasmid can express green fluorescent protein and resistant blasticidin gene (Blasticidin gene) in embryonic stem cells. Then 8 g of the modified Plenti6.0 linearized vector (FIG. 9) were transfected by electroporation in 10 6
- Example 2 was haploid embryonic stem cell lines of mouse and rat haploid embryonic stem cell lines, by 10 g / ml Blasticidin After screening for sputum, the resistant green fluorescence was picked (as shown in Figure 10, which is the result of the mouse transgenic haploid embryonic stem cell line, the results of the rat transgenic haploid embryonic stem cell line are not shown). system.
- the obtained GFP-positive cell line was screened by flow cytometry by Hoechst 33342 live cell staining (6) to obtain an animal transgenic embryonic stem cell line which maintained both haploid and exogenous GFP protein.
- the results are shown in Fig. 11.
- the results of the rat transgenic haploid embryonic stem cell line were not shown.
- Example 2 The orphan male embryonic stem cell line obtained in Example 2 was replaced with the animal transgenic embryonic stem cell line obtained in Example 6 in accordance with the method and conditions described in Example 4, thereby obtaining transgenic mice and transgenic rats.
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Abstract
一种动物胚胎干细胞系及其制备方法和应用,其中动物胚胎干细胞系为小鼠单倍体胚胎干细胞系或大鼠单倍体胚胎干细胞系,所述动物胚胎干细胞能够用于制备转基因动物及细胞分化。动物胚胎干细胞系的制备方法包括以下步骤:构建成孤雄单倍体胚胎;将制得的孤雄单倍体胚胎培养成桑椹胚;或将制得的孤雄单倍体胚胎培养成囊胚;将桑椹胚或囊胚培养成原代孤雄单倍体胚胎干细胞;再将所述原代孤雄单倍体胚胎干细胞传代培养,并通过流式细胞仪分选,制得孤雄单倍体胚胎干细胞系。
Description
一种动物胚胎干细胞系及其制备方法和应用 技术领域
本发明涉及一种干细胞及其制备方法和应用,尤其涉及一种动物胚胎干 细胞系及其制备方法和应用, 属于细胞生物学和发育生物学技术领域。 背景技术
转基因动物对于现代医学及生物学研究提供了重要的研究模型,但由哺 乳动物获得转基因动物仍然非常困难。
在现有技术中, 转基因动物主要是通过原核显微注射法, 或者利用胚胎 干细胞和同源重组技术, 对动物进行转基因而获得。 原核显微注射法是将外 源的目的片段导入到雄原核内, 此法一直都被认为是转基因动物研制的经典 方案, 但其有很大的局限性, 突出表现为外源基因整合位点和基因拷贝数的 高度不确定性, 由此往往引起动物后代表型差别很大; 利用胚胎干细胞和同 源重组技术, 通过胚胎干细胞种系嵌合的方案, 可以实现定点敲入或敲除的 基因打靶, 但其局限性在于获得纯种转基因动物的周期长, 且效率不高。
并且, 对于啮齿类之外的哺乳动物, 如非人灵长类, 缺乏具有种系嵌合 的胚胎干细胞, 从而缺乏可以传递遗传修饰的载体, 因此很难获得转基因动 物。
因而, 研究设计一种能够高效快速、 应用范围广地制备转基因动物的方 法, 极大地推动转基因动物生产技术在全世界范围内的推广和应用, 成为一 种必要。 发明内容
因此, 本发明的目的是针对目前转基因动物生产技术效率低, 不能广范 围地推广和应用的不足, 提供一种动物胚胎干细胞系及其制备方法和应用, 就可以快速、 直接、 高效地获得转基因动物。 本发明还开创了一种生产转基 因动物的快速而直接的方法, 使疾病模型的转基因动物的生产更加简捷。
针对上述目的, 本发明提供的技术方案如下:
一方面, 本发明提供一种小鼠单倍体胚胎干细胞系, 所述细胞系是保藏 号为 CGMCC No. 6037的孤雄单倍体胚胎干细胞系 N-1-1 (该细胞系的分类
命名为小鼠单倍体胚胎干细胞, 并于 2012年 4月 19 日保藏于中国微生物 菌种保藏管理委员会普通微生物中心 (CGMCC ) , 地址: 北京市朝阳区 北辰西路 1号院 3号, 中国科学院微生物研究所; 邮编: 100101 ) 。
优选地, 所述小鼠单倍体胚胎干细胞系可分化为三个胚层的细胞, 更优 选地, 所述三个胚层的细胞为脑室、 肌肉和腺体。
另一方面, 本发明提供一种大鼠单倍体胚胎干细胞系, 所述细胞系是保 藏号为 CGMCC No. 6038的孤雄单倍体胚胎干细胞系 HD2-2 (该细胞系的 分类命名为大鼠单倍体胚胎干细胞, 并于 2012年 4月 19日保藏于中国微 生物菌种保藏管理委员会普通微生物中心 (CGMCC ) , 地址: 北京市朝 阳区北辰西路 1号院 3号, 中国科学院微生物研究所; 邮编: 100101 ) 。
优选地, 所述大鼠单倍体胚胎干细胞系可分化为三个胚层的细胞, 更优 选地, 所述三个胚层的细胞为脑室、 肌肉和腺体。
还一方面, 本发明提供一种动物转基因胚胎干细胞系, 所述细胞系是通 过将 EFl a启动子连接的 GFP基因的质粒转染于本发明所述的小鼠单倍体 胚胎干细胞系或本发明所述的大鼠单倍体胚胎干细胞系而获得。
再一方面, 本发明提供一种动物胚胎干细胞系的制备方法, 包括以下步 骤:
1 )将核移植的动物精子细胞注入核移植的动物卵母细胞, 或将核移植 的动物精子细胞注入动物卵母细胞后去雌核, 然后再吸出卵母细胞的纺锤 体, 构建成孤雄单倍体胚月台 ( Androgenetic haploid embryonic stem cells, ahESCs );
2 )将步骤 1 )制得的孤雄单倍体胚胎培养成桑葚胎; 或将步骤 1 )制得 的孤雄单倍体胚胎培养成嚢胚;
3 )将步骤 2 )培养成的桑葚培或嚢胚先接种于饲养层细胞上, 再接种于 胚胎干细胞培养液中, 制得原代孤雄单倍体胚胎干细胞;
4 )将所述原代孤雄单倍体胚胎干细胞传代培养至 4〜5代时, 通过流式 细胞仪分选出孤雄单倍体胚胎干细胞;
5 )重复步骤 4 ), 直至获得单倍体比例为 85%〜100%的孤雄单倍体动物 胚胎干细胞系。
优选地, 在步骤 1 ) 中, 所述精子细胞为去尾后的精子头, 于 M2操作 液中将去尾后的精子头注入卵母细胞中; 优选地, 所述动物精子细胞和动物 卵母细胞来源于小鼠或大鼠。
优选地, 在步骤 1 ) 中, 所述雌核通过 HCG激素超排处理 20小时后去 除。
优选地, 在步骤 2 ) 中, 将孤雄单倍体胚胎在含有 5 μ g/ml细胞松弛素 的 KSOM-AA培养液中激活 5-6h后, 再将激活后的孤雄单倍体胚胎转移至 不含细胞松弛素的 KSOM-AA培养液中进行体外培养 72小时形成桑葚胎。
优选地, 在步骤 2 ) 中, 将孤雄单倍体胚胎移植入 0.5dpc的假孕动物输 卵管中, 96h后形成嚢胚。
优选地, 在步骤 3 ) 中, 所述饲养层细胞由胚胎成纤维细胞制得, 所述 胚胎干细胞培养液包括 N2B27培养液和添加物, 优选地, 当精子细胞和卵 母细胞来源于小鼠时, 所述添加物包括 Knockout血清替代物 ( KOSR )、 白 血病抑制因子、 MEK抑制因子、 GSK3 β抑制因子和细胞周期蛋白 Ρ53的抑 制因子; 或
优选地, 当精子细胞和卵母细胞来源于大鼠时, 所述添加物包括 Υ27632, A83-01、 CHIR99021和 PD0325901 ;
更优选地, 所述添加物还包括生长因子, 最优选地, 所述生长因子为大 鼠生长因子或小鼠生长因子。
又一方面, 本发明提供一种转基因动物的制备方法, 包括以下步骤: 将 EF1 α启动子连接的 GFP基因的质粒转染于本发明所述的动物胚胎干细胞系 的制备方法制得的动物胚胎干细胞系, 制得动物转基因胚胎干细胞系, 再将 所述动物转基因胚胎干细胞系注射入卵母细胞胞浆中获得重构胚,再将重构 胚移植入假孕动物体内, 假孕发育后, 获得转基因动物, 优选地, 将重构胚 移植入 0.5dpc的假孕动物输卵管内; 优选地, 所述假孕动物为小鼠或大鼠。
优选地, 将所述动物转基因胚胎干细胞系通过显微操作注射入预激活的 ΜΠ期的卵母细胞胞质中得到重构胚, 再将重构胚激活后, 体外培养至为 2 倍体;
优选地, 所述 ΜΠ期的卵母细胞通过 SrCl2预激活 20分钟;
优选地, 当所述假孕动物为小鼠时, 所述重构胚用含 SrCl2的 CZB激活 液激活 3小时; 或
当所述 4叚孕动物为大鼠时, 所述重构胚用 150μΜ 的丁 内 酯 I(Butyrolactone I, BLI)激活液(购自 BIOMOL公司 )激活 3小时;
优选地, 所述重构胚体外培养 24小时至为 2倍体。
优选地, 所述动物转基因胚胎干细胞系注射入卵母细胞胞浆中前还包括
将细胞系筛选为 G0/G1 期细胞系或核中期细胞系的步骤, 优选地, 所述
G0/G1期细胞系通过先将所述的动物转基因胚胎干细胞系进行活体染色, 再 通过流式细胞仪筛选得到; 所述核中期细胞系通过将动物转基因胚胎干细胞 系用秋水仙素预处理 3小时或用诺考达唑预处理 10小时得到。
还一方面, 本发明提供一种本发明所述的小鼠单倍体胚胎干细胞系或本 发明所述的大鼠单倍体胚胎干细胞系或本发明所述的动物胚胎干细胞系的 制备方法制备的动物胚胎干细胞系在制备转基因动物试剂盒中的应用,优选 地, 所述转基因动物试剂盒为转基因小鼠试剂盒或转基因动物试剂盒。
再一方面, 本发明提供一种本发明所述的小鼠单倍体胚胎干细胞系或发 明所述的大鼠单倍体胚胎干细胞系或发明所述的动物胚胎干细胞系的制备 方法制备的动物胚胎干细胞系在制备用于分化为三个胚层的细胞的试剂盒 中的应用, 优选地, 所述三个胚层的细胞为脑室、 肌肉和腺体。
又一方面, 本发明提供一种转基因动物试剂盒或细胞分化试剂盒, 包括 本发明所述的小鼠单倍体胚胎干细胞系或本发明所述的大鼠单倍体胚胎干 细胞系或本发明所述的动物胚胎干细胞系的制备方法制备的动物胚胎干细 胞系。 本发明所述的孤雄单倍体动物胚胎干细胞系不仅具有胚胎干细胞的基 本特性, 而且其能够使卵母细胞受精,进而发育形成完整的动物个体。故而, 从制作转基因动物的角度来讲, 可以对 ahESCs进行基因打靶, 然后通过卵 母细胞胞浆注射的途径获得带有特定基因修饰的动物后代。本发明涉及的利 用孤雄单倍体胚胎干细胞系研制转基因动物的方法,一方面,可以对 ahESCs 进行高效地定点打靶, 并且利用干细胞快速自我更新的特点大量扩增转基因 单倍体干细胞; 另一方面, 利用 ahESCs能够使卵母细胞体外受精发育成动 物个体的能力, 将基因改造的遗传特征遗传至下一代, 并通过杂交一代的自 交, 就可以快速、 直接、 高效地获得转基因动物, 无需胚胎干细胞种系遗传, 这大大缩短了获得纯种转基因动物的周期。 研究表明, 孤雄单倍体胚胎干细 胞系在基因表达上表现出与二倍体胚胎干细胞相似的特征, 而与圓形精子细 胞差异很大。 所以, 本发明无论是对于基础科学研究, 还是对于研究新型的 转基因动物制作技术, 都具有巨大的理论和应用价值。 本发明通过构建小鼠 和大鼠两类啮齿类动物的 ahESCs, 分别使其能够遗传至下一代, 不仅为开 创新型辅助生殖技术奠定了基础, 同时还开创了一种生产转基因动物的快速
而直接的方法, 使疾病模型的转基因动物的生产更加简捷、 方便。 从转基因 单倍体细胞直接到转基因动物的新方法, 不仅可以帮助大动物的优育优生, 繁育优质新品种, 同时还能从细胞的角度, 改善父源遗传疾病, 甚至加速物 种的进化。 附图的简要说明
以下, 结合附图来详细说明本发明的实施例, 其中:
图 1显示本发明所述的单倍体胚胎干细胞系通过流式细胞仪进行单倍 体分选的试验结果, 图中, P2表示 G0/G1期的单倍体胚胎干细胞系; 图 2显示小鼠的单倍体胚胎细胞;
图 3显示本发明所述的小鼠单倍体胚胎干细胞系;
图 4显示本发明所述的孤雄单倍体胚胎干细胞系的核型; 图 5显示本发明所述的孤雄单倍体胚胎干细胞表达不同多能性的分 子标记通过免疫荧光染色的试验结果, 其中, 图 5A为表达 Oct3/4通 过免疫荧光染色的试验结果; 图 5B为表达 Nanog通过免疫荧光染色的 试验结果; 图 5C为表达 Sox2通过免疫荧光染色的试验结果; 图 5D为 表达 SSEA- 1通过免疫荧光染色的试验结果;
图 6 显示本发明所述的孤雄单倍体胚胎干细胞系具有三胚层 的分化潜能, 其中, 图 6A为本发明所述的孤雄单倍体胚胎干细胞 系分化形成的脑室; 图 6B为本发明所述的孤雄单倍体胚胎干细胞 系分化形成的肌肉; 图 6C为本发明所述的孤雄单倍体胚胎干细胞 系分化形成的腺体;
图 7 显示本发明所述的孤雄单倍体胚胎干细胞系注入卵母胞 浆细胞后得到的单倍体后代;
图 8显示本发明所述的单倍体后代小鼠胎儿的 SSLP遗传学分析结果, 其中,图 8A为 PCR扩增小鼠样品中 D7Mit44的电泳结果; 图 8B为 PCR 扩增小鼠样品中 D8Mit94的电泳结果; 图中 1表示 DNA标记, 2表 示 CD- I 小鼠尾尖细胞样品的 PCR产物, 3表示 C57BL/6小鼠尾尖 细胞样品的 PCR产物, 4为 DB A/2小鼠尾尖细胞样品的 PCR产物, 5为 129Sv/Jae小鼠尾尖细胞样品的 PCR产物, 6为本发明所述的 孤雄单倍体胚胎干细胞系 (N- 1 - 1 )样品的 PCR产物, 7为单倍体后 代小鼠胎儿尾尖细胞样品的 PCR产物;
图 9显示改造后的 Plenti6.0线性化载体的图谱;
图 10 显示本发明所述的小鼠转基因胚胎干细胞系的绿色荧光 蛋白显色图;
图 11 显示本发明所述的小鼠转基因胚胎干细胞系通过流式细胞仪 进行单倍体分选的试验结果, 图中, P2表示 G0/G1 期和核中期的单倍体 胚胎干细胞系。
本发明的生物材料的保藏信息:
保藏号为 CGMCC No. 6037的小鼠胚胎干细胞, 该细胞系的分类命名 为小鼠单倍体胚胎干细胞, 并于 2012年 4月 19日保藏于中国微生物菌种 保藏管理委员会普通微生物中心 (CGMCC ) , 地址: 北京市朝阳区北辰 西路 1号院 3号, 中国科学院微生物研究所; 邮编: 100101。
保藏号为 CGMCC No. 6038的大鼠胚胎干细胞, 该细胞系的分类命名 为大鼠单倍体胚胎干细胞, 并于 2012年 4月 19日保藏于中国微生物菌种 保藏管理委员会普通微生物中心 (CGMCC ) , 地址: 北京市朝阳区北辰 西路 1号院 3号, 中国科学院微生物研究所; 邮编: 100101。 实施发明的最佳方式
可以理解的是, 在此描述的特定实施方式通过举例的方式来表示, 其并 不作为对本发明的限制。 在不偏离本发明范围的情况下, 本发明的主要特征 可以用于各种实施方式。 本领域的技术人员将会意识到或能够确认, 仅仅使 用常规实验, 许多等同物都能应用于本文所描述的特定步骤中。 这些等同物 被认为处在本发明的范围之内, 并且被权利要求所覆盖。 除非特别说明, 以下实施例中所用的试剂均可从常规渠道获得, 所用的 方法均为常规试 ^全方法。
以下实施例中所用的试验材料 CD-I ( ICR )、 C57BL/6、 DBA/2、 129Sv/Jae 和 SCID品系的小鼠, DA、 SD品系的大鼠均购自北京维通利华实验动物技 术有限公司。
B6D2F1品系的小鼠由购买的 C57BL/6和 DBA/2品系的小鼠交配所得, 小鼠胚胎成纤维细胞购自中国科学院动物研究所。 M2操作液(购自 Sigma 公司) , KSOM-AA培养液和 CZB激活液的配方分别如下:
表 1 配制 lOO ml KSOM (培养液)所需的组分及含量
注: *制作微滴用, 因计划使用量可适当增减配液的总体积(储存时间不可过两周) 实施例 1构建孤雄单倍体胚胎
釆用两种不同的方法获得孤雄单倍体胚胎, 这两者方法在大小鼠中都可
以用于构建单倍体胚胎, 此处分别阐述。
小鼠单倍体胚胎: 通过 Qi Zh0U开创的"一步法 "核移植技术进行孤雄单 倍体胚胎重构 (1)。 具体如下, 取用 8周龄的 129Sv/Jae雄鼠以断颈法处死, 取其输精管内的精子。 将精子在超声波震荡仪中断去尾部, 剩下精子头部, 这些精子头部作为核移植的供体细胞。核移植的卵母细胞取自 8周龄的接受 过超排处理的 B6D2F 1雌鼠。 在 piezo压电式显微操作仪下, 于 M2操作液 中进行显啟操作, 将预处理好的精子头部注入到卵母细胞中, 同时吸出卵母 细胞的纺锤体, 重构好的胚胎称为孤雄单倍体胚胎( Androgenetic haploid embryo, AHE ) 。 核移植操作后, 重构单倍体胚胎在含有 5 g/ml细胞松弛 素(CB )的 KSOM-AA培养液中激活(精子本身成分即可激活卵母细胞) , CB的目的是防止极体排出, 以确保胚胎基因组的单倍体特性。 激活 5〜6小 时后, 将单倍体胚胎转移入不含 CB的 KSOM-AA培养液中进行体外培养, 培养箱的条件为 37°C , 5% C02。 孤雄单倍体胚胎在体外培养 72小时后, 部 分胚胎发育至桑葚胚, 这些桑葚胚种植入胚胎干细胞培养液中可建立单倍体 胚胎干细胞系。
大鼠单倍体胚胎:孤雄单倍体重构釆取受精卵去雌原核的方法。具体为, 体内冲取 DA、 SD的品系杂交受精卵, 在 HCG激素超排处理 20小时后, 进行去原核操作。 重构后的孤雄大鼠单倍体胚胎移植入 0.5 dpc的假孕母鼠 输卵管中, 96小时后冲取大鼠孤雄单倍体嚢胚。 实施例 2孤雄单倍体胚胎干细胞系 ( ahESCs ) 的建立
利用重构的 AHE桑葚胚, 高效的建立 ahESC细胞系。 建系初期, 将单 倍体胚胎接种在饲养层细胞上, 所述饲养层细胞是利用小鼠胚胎成纤维细胞 制备的。 建立单倍体胚胎干细胞系的培养液由 N2B27基础培养液 (GIBCO) 及各种添加物所组成,其中 N2B27基础培养液参照 Ying QL等的报道 (2) , 添 加物包括 Knockout血清替代物 (KOSR ) ( GIBCO ) 、 白血病抑制因子 (Millipore), MEK抑制因子 (Stemgent)、 GSK3 抑制因子 (Stemgent)、 细胞周 期蛋白 P53的抑制因子 (Calbiochem)等。 胚胎接种后 6天左右挑克隆, 挑克 隆时用 0.25%胰酶进行消化, 消化后继续培养建立单倍体干细胞系, 此时细 胞的代次记为原代孤雄单倍体胚胎干细胞(P1 )。 每隔 2〜3天, 将单倍体胚 胎干细胞传代一次。 在单倍体细胞传至第 4〜5代时, 通过细胞流式纯化细胞 系中的单倍体细胞。流式筛选的方法主要参照 Elling U等人的单倍体纯化办
法 (3) , 本实 3全釆用 ^gml"1 Hoechst33342和 50μΜ Verapamil ( Sigma )处理细 胞 30分钟, 然后进行单倍体细胞分选 (3)。 在细胞培养的过程, 每隔 4〜5代对 细胞系进行单倍体纯化, 获得长期稳定的孤雄单倍体胚胎干细胞系, 即小鼠 单倍体胚胎干细胞系, 结果如图 1所示, 最后得到单倍体比例为 85%以上的 单倍体胚胎干细胞系。 通过活细胞工作站(莱卡)观察该小鼠单倍体胚胎干 细胞系 (即保藏号为 CGMCC No.6037的小鼠单倍体胚胎干细胞系) , 结果 如图 3所示, 该小鼠单倍体胚胎干细胞系具有与二倍体小鼠干细胞(ESCs ) 相似的克隆形态。
大鼠单倍体胚胎干细胞系的建系体系与小鼠相同, 不同的是在小鼠的培 养基的基础上将小鼠生长因子更换为大鼠生长因子, 并将添加物更改为 Y27632 ( MERCK ) 、 A83-01 ( Stemgent ) 、 CHIR99021 ( Stemgent )和 PD0325901(Stemgent)四种生长因子,得到大鼠单倍体胚胎干细胞系, 即保藏 号为 CGMCC No.6038的大鼠单倍体胚胎干细胞系, 具体结果未显示。 实施例 3 ¾ 单倍体干细胞系的干细胞功能^
利用 G-带核型的方法 (3)检测实施例 2制得的孤雄单倍体胚胎干细胞系即 小鼠单倍体胚胎干细胞系, 结果如图 4所示, 其染色体核型为 19+X。
纯化后的孤雄单倍体胚胎干细胞系仍然表达多能性的分子标记, 如 Oct3/4, Nanog, Sox2, SSEA-1等 (6), 首先将细胞用 4%的多聚曱醛固定 20分 钟, 随后加 0.5%的 Triton X-100处理 30分钟, 添加一抗: 抗 - Oct3/4
( anti-Oct3/4 ) (Santa Cruz), 抗 -Sox2 ( anti-Sox2 ) (Santa Cruz), 抗 -SSEA1 ( anti-SSEAl )和抗 -Nanog ( anti-Nanog ) (Millipore) 在 4°C冰箱孵育过夜, 随后用二抗: AlexaFlur 488连接的二抗 ( AlexaFlur 488-conjugated secondary antibody, sigma ) 室温孵育 1小时之后在激光共聚焦显微镜下观察(蔡司, LSM 780 META). (见图 5 ) 。
将 1 X 107个孤雄单倍体胚胎干细胞系注射入 6周龄的免疫缺陷 SCID品 系的雄鼠大腿内侧皮下, 三周后长出畸胎瘤, 将畸胎瘤用 4%的多聚曱醛固 定, 石蜡包埋切片, 进行组织学分析发现 (6), 该干细胞系在体外可分化形成 脑室、 肌肉和腺体三个胚层的细胞, 具有三胚层的分化潜能(见图 6, 小鼠 孤雄单倍体胚胎干细胞系的结果, 大鼠孤雄单倍体胚胎干细胞系的结果未显 示) 。
这些结果表明我们所建立的孤雄单倍体胚胎干细胞系, 与二倍体胚胎干 细胞一样, 具有胚胎干细胞的基本特征。
实施例 4孤雄单倍体胚胎干细胞系注射入卵胞浆中发育成后代
借鉴卵母细胞胞浆内注射 ( ICSI )实验和圓形精子卵胞浆内注射 ( ROSI ) 实验 (4), 将实施例 2得到的孤雄胚胎干细胞系注射入卵母细胞胞浆中, 通过 人工激活, 并将激活后的胚胎移植入假孕鼠体内, 可以获得该胚胎发育而来 的子代, 此法我们定义为单倍体胚胎干细胞系卵胞浆内注射
( Introcytoplasmicandrogenetic haploid ES cell injection, ICAI )。 单倍体供体 细胞分为两类: G0/G1期细胞和核中期细胞, G0/G1期细胞通过 Hoechst33342 活体染色, 并通过流式细胞仪精确地筛选得到, 而核中期细胞则通过秋水仙 素预处理 3小时或诺考达唑预处理 10小时得到。 两类供体细胞分别通过显 微操作注射入 SrCl2预激活 20分钟的 ΜΠ期的 CD-I系小鼠的卵母细胞胞质 中, 再通过含 SrCl2的 CZB激活液进行激活 3小时。 激活后的重构胚转移入 KSOM-AA培养液中于 37°C , 5%C02进行体外培养。 体外培养 24小时后, 统计卵裂率, 将所有的 2倍体细胞胚胎移植入 0.5dpc ( 0.5天) 的假孕小鼠 输卵管中。假孕小鼠发育 19.5天后分娩或剖腹产,得到单倍体后代小鼠胎儿, 结果如图 7所示。 大鼠的卵母细胞胞浆内注射过程和小鼠相似, 但是激活液 改为终浓度为 150μΜ的丁内酯 I激活液( Butyrolactonel激活液 ) , 孕期为 21.5天, 能够得到健康后代。
部分的 ICAI出生的小鼠胎儿能存活, 并能成长至性成熟; 将其与野生 型小鼠交配, 可以繁育下一代, 说明单倍体胚胎干细胞系 ICAI动物具有正 常的繁殖功能。 实施例 5单倍体后代小鼠胎儿的 SSLP遗传学分析
通过微卫星标记分子 D7Mit44、 D8Mit94(5)进行 SSLP遗传学分析, 鉴定 孤雄单倍体胚胎干细胞系与其 ICAI得到的后代小鼠。
取 CD-I ( ICR ) , C57BL/6, DBA/2、 129Sv/Jae品系的小鼠的尾尖细胞, 实施例 2制得的单倍体小鼠单倍体胚胎干细胞系, 实施例 4获得的单倍体小 鼠胎儿的尾尖细胞, 提取 DNA, 进行 PCR扩增, 引物来自
MGI(htt ://www. informatics.j ax. org/searches/probe_form. shtml) , 由生工生物 (上海)有限公司合成, 具体如下述表 3所示:
表 3 引物
扩增条件为 94°C 30秒, 55°C 30秒, 72°C 30秒, 扩增 35个循环, 进 行 PCR扩增, 并将 PCR产物电泳后, 进行显色分析, 图 8A为扩增样品中 D7Mit44的试验结果, 图 8B为扩增样品中 D8Mit94的试验结果, 表明后代 小鼠同时具有孤雄单倍体胚胎干细胞系 (129Sv/Jae )和卵母细胞(CD-I ( ICR ) )两套基因组, 说明孤雄单倍体胚胎干细胞系 ICAI后代小鼠确实来 源于相对应的单倍体胚胎干细胞。 实施例 6对 单倍体胚胎干细胞系进行转基因修饰
以 GFP荧光蛋白为例对孤雄单倍体胚胎干细胞系进行转基因标记。 按 常规的方法将 Plenti6.0载体(购买自 Invitrogen公司 )携带的 CMV启动子 替换为可以在胚胎干细胞中表达的 EFla启动子 (SEQ ID NO:5 ) (上游引 物: GAATTGGCTCCGGTGCCCGTCAGT ( SEQ ID NO:6 ) ; 下游引物: AATTCCTCACGACACCTGAAATGG ( SEQ ID NO:7 ) , 由生工生物(上海) 有限公司合成; 扩增条件: 95 °C 30秒; 59°C 30秒; 72°C 1分 30秒; 35 循环) , 并在其后多克隆位点处连接上绿色荧光蛋白基因 (GFP ) ( SEQ ID NO:8 ) (上游引物: ATGGTGAGCAAGGGCGAGGAGC ( SEQ ID NO:9 ) ; 下游引物: TTACTTGTACAGCTCGTCCATG ( SEQ ID NO: 10 ) , 由生工生 物 (上海)有限公司合成; 扩增条件: 95 °C 30秒; 61 °C 30秒; 72°C 1分 30秒; 35循环) , 还连接有抗性选择基因抗性杀稻瘟菌素基因 (Blasticidin 基因) , 进行载体的构建。 改造后的质粒可以在胚胎干细胞中表达表达绿色 荧光蛋白和抗性杀稻瘟菌素基因 (Blasticidin基因) 。 然后将 8 g改造后的
Plenti6.0线性化载体(图 9 )通过电转分别转染于 106个实施例 2制得的小 鼠单倍体胚胎干细胞系和大鼠单倍体胚胎干细胞系,经 10 g/ml杀稻瘟菌素 筛选后挑取抗性绿色荧光(如图 10所示, 为小鼠转基因单倍体胚胎干细胞 系的结果, 大鼠转基因单倍体胚胎干细胞系的结果未显示 )表达克隆扩增成 系。 获得的 GFP阳性细胞系通过 Hoechst33342活细胞染色法 (6), 通过流式 细胞仪进行筛选得到既维持单倍体又表达外源 GFP蛋白的动物转基因胚胎 干细胞系, 结果如图 1 1所示, 为小鼠转基因单倍体胚胎干细胞系的结果, 大鼠转基因单倍体胚胎干细胞系的结果未显示。 实施例 7转基因动物的获得
按照实施例 4描述的方法和条件将实施例 2得到的孤雄胚胎干细胞系替 换为实施例 6获得的动物转基因胚胎干细胞系,从而获得转基因小鼠和转基 因大鼠。
参考文献
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4. H. Ohta, Y. Sakaide, T. Wakayama, Functional analysis of male mouse haploid germ cells of various differentiation stages: early and late round spermatids are functionally equivalent in producing progeny. Biology of reproduction^, 51 1 (Mar, 2009).
5. X. Y. Zhao et al, iPS cells produce viable mice through tetraploid complementation. NatureA6\, 86 (Sep 3, 2009).
6. Zhao XY*, Li W, Lv Z, Zeng FY5", Zhou (f . (2010) Production of mice using iPS cells and tetraploid complementation. Nat Protoc. 5(5):963-71.
Claims
1. 一种小鼠单倍体胚胎干细胞系, 所述细胞系是保藏号为 CGMCC No.6037的孤雄单倍体胚胎干细胞系, 优选地, 所述小鼠单倍体胚胎干细胞 系分化为三个胚层的细胞, 更优选地, 所述三个胚层的细胞为脑室、 肌肉 和腺体。
2. 一种大鼠单倍体胚胎干细胞系, 所述细胞系是保藏号为 CGMCC No.6038的孤雄单倍体胚胎干细胞系, 优选地, 所述大鼠单倍体胚胎干细胞 系分化为三个胚层的细胞, 更优选地, 所述三个胚层的细胞为脑室、 肌肉 和腺体。
3. 一种动物转基因胚胎干细胞系,所述细胞系是通过将 EF1 α启动子连 接的 GFP基因的质粒转染于权利要求 1所述的小鼠单倍体胚胎干细胞系或 权利要求 2所述的大鼠单倍体胚胎干细胞系而获得。
4. 一种动物胚胎干细胞系的制备方法, 包括以下步骤:
1 )将核移植的动物精子细胞注入核移植的动物卵母细胞, 或将核移植 的动物精子细胞注入动物卵母细胞后去雌核, 然后再吸出卵母细胞的纺锤 体, 构建成孤雄单倍体胚胎;
2 )将步骤 1 )制得的孤雄单倍体胚胎培养成桑葚胎; 或将步骤 1 )制得 的孤雄单倍体胚胎培养成嚢胚;
3 )将步骤 2 )培养成的桑葚胚或嚢胚先接种于饲养层细胞上, 再接种于 胚胎干细胞培养液中, 制得原代孤雄单倍体胚胎干细胞;
4 )将所述原代孤雄单倍体胚胎干细胞传代培养至 4〜5代时, 通过流式 细胞仪分选出孤雄单倍体胚胎干细胞;
5 )重复步骤 4 ), 直至获得单倍体比例为 85%〜100%的孤雄单倍体动物 胚胎干细胞系。
5. 根据权利要求 4所述的动物胚胎干细胞系的制备方法, 其特征在于, 在步骤 1 ) 中, 所述精子细胞为去尾后的精子头, 于 Μ2操作液中将去尾后 的精子头注入卵母细胞中; 优选地, 所述动物精子细胞和卵母细胞来源于小 鼠或大鼠; 优选地, 所述雌核通过 HCG激素超排处理 20小时后去除。
6. 根据权利要求 4或 5所述的动物胚胎干细胞系的制备方法,其特征在 于,在步骤 2 )中,将孤雄单倍体胚胎在含有 5 μ g/ml细胞松弛素的 KSOM-AA 培养液中激活 5〜- 6h后, 再将激活后的孤雄单倍体胚胎转移至不含细胞松弛 素的 KSOM-AA培养液中进行体外培养 72小时 , 形成桑葚胎。
7. 根据权利要求 4至 6中任一项所述的动物胚胎干细胞系的制备方法, 其特征在于, 在步骤 2 ) 中, 将孤雄单倍体胚胎移植入 0.5dpc的假孕雌性动 物输卵管中, 96h后形成嚢胚。
8. 根据权利要求 4至 7中任一项所述的动物胚胎干细胞系的制备方法, 其特征在于, 在步骤 3 ) 中, 所述饲养层细胞由胚胎成纤维细胞制得, 所述 胚胎干细胞培养液包括 N2B27培养液和添加物, 优选地, 当精子细胞和卵 母细胞来源于小鼠时,所述添加物包括血清替代物 KOSR、白血病抑制因子、 MEK抑制因子、 GSK3 β抑制因子和细胞周期蛋白 P53的抑制因子; 或
优选地, 当精子细胞和卵母细胞来源于大鼠时, 所述添加物包括
Υ27632, A83-01、 CHIR99021和 PD0325901 ;
更优选地, 所述添加物还包括生长因子, 最优选地, 所述生长因子为大 鼠生长因子或小鼠生长因子。
9. 一种转基因动物的制备方法, 包括以下步骤, 将 EFl a启动子连接的 GFP基因的质粒转染于权利要求 4至 8中任一项所述的制备方法制得的动物 胚胎干细胞系, 制得动物转基因胚胎干细胞系, 再将所述动物转基因胚胎干 细胞系注射入卵母细胞胞浆中获得重构胚, 再将重构胚移植入假孕动物体 内, 假孕发育后, 获得转基因动物, 优选地, 将重构胚移植入 0.5dpc的假孕 动物输卵管内; 优选地, 所述假孕动物为小鼠或大鼠。
10. 根据权利要求 9所述的转基因动物的制备方法, 其特征在于, 将所 述动物转基因胚胎干细胞系通过显微操作注射入预激活的 ΜΠ 期的卵母细 胞胞质中得到重构胚, 再将重构胚激活后, 体外培养至为 2倍体;
优选地, 所述 ΜΠ期的卵母细胞通过 SrCl2预激活 20分钟;
优选地, 当所述假孕动物为小鼠时, 所述重构胚用含 SrCl2的 CZB激活 液激活 3小时; 或
当所述^ _孕动物为大鼠时, 所述重构胚用 150μΜ的丁内酯激活液激活 3小时;
优选地, 体外培养 24小时至为 2倍体。
11. 根据权利要求 9或 10所述的转基因动物的制备方法, 其特征在于, 所述动物转基因胚胎干细胞系注射入卵母细胞胞浆中前还包括将细胞系筛 选为 G0/G1期细胞系或核中期细胞系的步骤, 优选地, 所述 G0/G1期细胞 系通过先将所述动物转基因胚胎干细胞系进行活体染色,再通过流式细胞仪 筛选得到; 所述核中期细胞系通过将动物转基因胚胎干细胞系用秋水仙素预 处理 3小时或用诺考达唑预处理 10小时得到。
12. 根据权利要求 1所述的小鼠单倍体胚胎干细胞系或 2所述的大鼠单 倍体胚胎干细胞系或权利要求 4至 8中任一项所述的动物胚胎干细胞的制备 方法制备的动物胚胎干细胞系在制备转基因动物试剂盒中的应用, 优选地, 所述转基因动物试剂盒为转基因小鼠试剂盒或转基因动物试剂盒。
13. 根据权利要求 1所述的小鼠单倍体胚胎干细胞系或 2所述的大鼠单 倍体胚胎干细胞系或权利要求 4至 8中任一项所述的动物胚胎干细胞的制备 方法制备的动物胚胎干细胞系在制备用于分化为三个胚层的细胞的试剂盒 中的应用, 优选地, 所述三个胚层的细胞为脑室、 肌肉和腺体。
14. 一种转基因动物试剂盒或细胞分化试剂盒, 包括权利要求 1所述的 小鼠单倍体胚胎干细胞系或 2所述的大鼠单倍体胚胎干细胞系或权利要求 4 至 8中任一项所述的动物胚胎干细胞的制备方法制备的动物胚胎干细胞系。
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