Generation of Mitochondrial-nuclear eXchange Mice via Pronuclear Transfer
通过原核移植生成线粒体-细胞核置换小鼠   

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Cancer Research
Oct 2015

 

Abstract

The mitochondrial paradigm for common disease proposes that mitochondrial DNA (mtDNA) sequence variation can contribute to disease susceptibility and progression. To test this concept, we developed the Mitochondrial-nuclear eXchange (MNX) model, in which isolated embryonic pronuclei from one strain of species are implanted into an enucleated embryo of a different strain of the same species (e.g., C57BL/6 and C3H/HeN, Mus musculus), generating a re-constructed zygote harboring nuclear and mitochondrial genomes from different strains. Two-cell embryos are transferred to the ostia of oviducts in CD-1 pseudopregnant mice and developed to term. Nuclear genotype and mtDNA haplotype are verified in offspring, and females selected as founders for desired MNX colonies. By utilizing MNX models, many new avenues for the in vivo study for mitochondrial and nuclear genetics, or mito-Mendelian genetics, are now possible.

Keywords: Mitochondria (线粒体), Mitochondrial DNA (线粒体DNA), Polymorphism (多态性), Crosstalk (串扰)

Background

The isolation of nuclear and mitochondrial genomes in MNX mice strains allows examination of pathomechanisms of dysfunctional bioenergetics such as cardiovascular disease (Fetterman et al., 2013; Grimsditch et al., 2000; Paigen et al., 1990; Wang et al., 2005), glucose tolerance (Freeman et al., 2006; Kaku et al., 1988) and fatty liver disease (Betancourt et al., 2014). This approach is distinct from conplastic (Yu et al., 2009) and xenomitochondrial (McKenzie et al., 2004) approaches in that MNX mice are generated directly with 100% of the desired nuclear and mtDNA complements from respective donor strains through nuclear transfer and thus do not require repeated back-crossings (as do conplastics) to generate animals having the desired genotype (Figure 1). Furthermore, MNX mice allow direct, unambiguous assessment of mtDNA contributions to disease since there is no complexity introduced by potential nuclear cross-over and combinational effects in the filial generations associated with standard backcrossing methods used to generate conplastic mice.

Materials and Reagents

  1. 26 gauge needle
  2. 1 or 3 ml syringe
  3. Microscope slides
  4. CellTram vario syringe
  5. 10 centimeter tissue culture dishes
  6. Female donor mice (3-4 weeks of age) of desired nuclear or mitochondrial genetic backgrounds
  7. Male breeders (proven) of matching nuclear background as donor females
  8. Female recipient mice (8-10 weeks of age)
  9. Vasectomized male mice (proven)
  10. Gonadotropin from pregnant mare serum (PMS) (Sigma-Aldrich, catalog number: G4877 )
    Note: This product has been discontinued.
  11. Human chorionic gonadotropin (HCG) (Sigma-Aldrich, catalog number: CG10 )
  12. M2 medium (Sigma-Aldrich, catalog number: M7167 )
  13. Cytochalasin B from Drechslera dematioidea (Sigma-Aldrich, catalog number: C6762 )
  14. Colcemid (Sigma-Aldrich, catalog number: D1925 )
  15. Embryo tested mineral oil (Sigma-Aldrich, catalog number: M8410 )
  16. Water for embryo transfer (Sigma-Aldrich, catalog number: W1503 )
  17. Restriction enzyme:
    1. BclI (New England Biolabs)
    2. PflFI/AspI (New England Biolabs)
  18. Dilution of PMS (see Recipes)
  19. Dilution of HCG (see Recipes)
  20. Dilution of cytochalasin B (see Recipes)
  21. Dilution of colcemid (see Recipes)

Equipment

  1. Piezoelectric drill (piezo drill) (Sutter Instrument, model: PrimeTech PMM-150FU )
  2. Electroporator (BTX The Electroporation Experts, model: ECM 630 )
  3. Micropipette puller (horizontal pipette puller) (Sutter Instrument, model: P-87 )
    The product P-87 has been discontinued and the available one is P-97 .
  4. Micropipette microforge (Defonbrune microforge with microscope head) (Leitz)
  5. Holding pipette puller (vertical pipette puller) (David Kopf Instrument, model: 720 )
  6. Microscope (Laborlux S Nomarski DIC) (Leica Microsystems)
  7. CellTram Vario syringe (Eppendorf)
  8. Benchtop incubator (Cook, model: MINC G20079 )
  9. 45 degree angled forceps (Fine Science Tools, catalog number: 11251-35 )
  10. Straight forceps (Fine Science Tools, catalog number: 11251-10 )
  11. Microdissecting spring scissors (Roboz Surgical Instrument, catalog number: RS-5650 )
  12. Mouth pipette

Procedure

  1. Creation of MNX mice


    Figure 1. Overall procedure schematic. MNX mice were produced by enucleating fertilized oocytes from 'Strain A' (C57BL/6) and 'Strain B' (C3H/HeN) mice. MNX nomenclature is indicated by strain nuclear (Strainn):strain mtDNA (Strainmt), e.g., mice with C3H nuclear DNA and C57 mtDNA are indicated by C3Hn:C57mt. Strain B pronuclei were transferred to enucleated Strain A oocytes yielding Strain Bn:Strain Amt (C3Hn:C57mt) oocytes, which were implanted into surrogate females to generate MNX progeny. The reciprocal process was followed to generate the Strain An:Strain Bmt (C57n:C3Hmt) oocytes.

    1. Female donor mice are injected intraperitoneally (IP) with PMS (5.0-7.5 IU, in a volume 0.2-0.3 ml) using a 26 gauge needle and a 1 or 3 ml syringe. Optimum dose and age of the mice can be strain specific, and should be determined via dose response curves with assessment of fertilized embryo production. Typically, female mice are injected ~noon on day 1.
    2. Approximately 48 h after (day 3) the PMS injection, donor mice receive an IP injection of HCG of the same dose as PMS. Immediately following injection, super-ovulated females are paired and mated with proven breeder males of the same nuclear genotype (matching the female, Figure 1); often copulation occurs within the first 1-2 h of mating. Female recipient mice positive for signs of proestrus by visual inspection are paired with proven vasectomized males to generate pseudopregnancy (Byers et al., 2012).
    3. Females are checked the following morning for vaginal plugs. The presence of a plug indicates successful mating, but does not necessarily indicate fertilization. The absence of a plug does not however confirm that successful breeding did not occur since facile identification of plugs can be strain dependent.
    4. Cumulus masses are harvested as previously described to produce single-celled embryos (Figures 2A and 2B) (Han et al., 2010).


      Figure 2. Collection of cumulus masses from oviducts. Oviducts (A) are dissected from superovulated females with vaginal plugs. The swollen ampulla (indicated by arrow) is nicked to allow the embryos and cumulus cells to be expelled (B).

    5. Pronuclear embryos are placed by mouth pipette in M2 medium containing cytochalasin B (5 μg/ml) and colcemid (0.1 μg/ml) at 37 °C for 5 min, and remain in a microinjection drop of M2 medium on a microscope slide at room temperature to prevent lysis during manipulation.
    6. A micropipette similar in size and shape to a beveled pipet used for embryonic stem cell injections (approximately 20 micron inner diameter) is used to remove both pronuclei of each embryo by applying slight pressure on the zona pellucida of the first embryo (Longenecker and Kulkarni, 2009). A high-intensity piezo pulse is applied only until the zona is ruptured (approximately 1-2 sec) and then turned off. The pipet is slowly advanced to each pronucleus (Figure 3), and with gentle suction applied to the needle using a CellTram vario syringe, the two pronuclei are aspirated and removed as a single unit (karyoplast, Figure 3). Appropriate needle size and application of suction only when the pronuclei are in contact with the needle minimizes uptake of cytoplasmic contents into the needle.
    7. The procedure is repeated on the reciprocal embryo, resulting in the pipet containing pronuclei from both strains (Figure 3).


      Figure 3. Isolation of pronuclei. (A) The pipet is slowly advanced to each pronucleus (indicated by black arrows), and with gentle suction applied to the needle using a CellTram vario syringe (B) the two pronuclei are aspirated and removed (C) as a single unit (karyoplast, K). ZP, zona pellucida.

    8. The isolated pronuclei from Strain A are implanted into the enucleated embryo of Strain B (Figure 4). The reciprocal karyoplasts from Strain B are placed into the enucleated Strain A embryos.


      Figure 4. Transfer of pronuclei. The karyoplasts (K) from one strain are then implanted into the enucleated embryos of the reciprocal strain. ZP, zona pellucida. (A) Insertion of pipet containing karyoplasts (B) Ejection of karyoplasts (C) Reconstructed embryo.

    9. Ten centimeter tissue culture dishes (Figure 5) are loaded with 30 μl microdrops of M2 medium and covered in mineral oil and each embryo is placed by mouth pipette into its own drop of media. Strain A and Strain B embryos are placed in separate dishes. An electrode is placed into the drop positioning the embryo between the two poles. A single 90 V pulse is applied to each re-constructed zygote and all zygotes are cultured overnight (Han et al., 2010).


      Figure 5. Embryo culture dish. Panels 1-8 are different drops of M2 medium and mineral oil containing reconstructed zygotes. Each drop may contain 1-2 zygotes.

    10. Two-cell embryos are transferred to the ostia of oviducts of 0.5 day pseudopregnant mice to term; approximately 20 embryos are transferred per recipient (Figure 6).


      Figure 6. Oviduct transfer of reconstituted embryos. Two cell embryos are placed into the oviduct of a pseudopregnant recipient mouse through the infundibular ostium by mouth pipet.

    11. Nuclear genotype and mtDNA haplotype are verified in offspring via nuclear SNP and complete mtDNA genome sequence analysis from ear or tail clips.

  2. MNX mouse colony establishment
    MNX mouse colonies are established using homoplasmic founder female MNX mice based upon PCR analysis specific for each potential mtDNA haplotype. mtDNA haplotype is initially confirmed by PCR detection methods developed specifically for each haplotype and homoplasmic females confirmed by sequencing. Desired homoplasmic (based upon PCR screening) females to be used as founders are identified in ~20%-30% of generated females.
    1. MNX females of verified mtDNA sequence and apparent homoplasmy (using ear or tail clip DNA) are crossed with males of the matching nuclear background to generate F1 litters.
    2. Multiple tissues from F1 progeny are tested to verify desired mtDNA haplotype/homoplasmy via PCR analysis designed specifically for diagnostic mtDNA SNP’s (Bayona-Bafaluy et al., 2003). For example, F1 C57n:C3Hmt and C3Hn:C57mt mice were haplogrouped in multiple tissues based upon a PCR detection method to interrogate the potential for heteroplasmy (Figure 7).
      PCR protocol:
      2x GoTaq MasterMix: 25 μl
      Primers: 5 μl each
      Nuclease free H2O: 12.5 μl
      DNA: 2.5 μl
      Thermocycling profile:
      95 °C for 2 min: 1 cycle
      95 °C for 30 sec; 57 °C for 1min; 72 °C for 30 sec: 35 cycles
      72 °C for 10 min: 1 cycle
      24 °C for infinity
      1. To screen for the ND3 mutation at bp 9461 (204 base pair amplicon):
        9461F: 5’-TTCCAATTAGTAGATTCTGAATAAACCCAGAAGAGAGTGAT-3’
        9461R: 5’-AAATTTTATTGAGAATGGTAGACG-3’
        Ten microliters of amplicon are digested with the restriction enzyme Bcli (10 U, New England Biolabs) in a 10 microliter reaction volume. The C3H mtDNA is cleaved into 166 bp and 38 bp fragments, while the C57 mtDNA remains uncut.
      2. To screen for the bp 9348 CO3 mutation (385 base pair amplicon):
        9348F: 5’- CGAAACCACATAAATCAAGCCC-3’
        9348R: 5’-CTCTCTTCTGGGTTTATTCAGA-3’
        Ten microliters of amplicon are digested using the Pflf1 (AspI, 10 U, New England Biolabs) restriction enzyme in a 20 microliter reaction volume. The C3H mtDNA remains uncut while C57 mtDNAs are digested into 274 bp and 111 bp fragments.


        Figure 7. Generation of Mitochondrial-nuclear eXchange (MNX) mice. Nuclear genotyping and mtDNA haplotyping of all originating founding females and F1 progeny was determined by nuclear SNP analysis of a panel of 38 distinguishing nuclear SNPs and complete sequencing of the mtDNA. After initial mtDNA haplotype verification by direct sequencing for founders, subsequent generations are haplotyped via restriction enzyme length polymorphism analysis using AspI and BclI which give patterns distinct for C3H and C57 mtDNAs. A. PCR products from C57 mtDNAs remain uncut (204 bp) whereas C3H mtDNAs are cleaved by BclI to yield 166 bp and 38 bp fragments. B. PCR products from C57 mtDNAs are cleaved by AspI to yield 274 bp and 111 bp fragments whereas C3H mtDNAs remain uncut (385 bp).

    3. MNX female founders that produced mice which appear homoplasmic in all tested tissues are then used as founding dams for each respective MNX colony.
    4. All progeny of each generation are mtDNA haplogrouped using PCR based techniques to verify maintenance of desired genetic background.

Data analysis

The original research paper detailing generation of MNX mice as well as analysis and replicate information is available online (Fetterman et al., 2013).

Recipes

  1. Dilution of PMS
    Add 20 ml embryo tested water to 1,000 IU bottle of lyophilized PMS to create 20 ml stocks at 50 IU/ml.
    Store at -20 °C for up to 3 months.
  2. Dilution of HCG
    Add 10 ml embryo tested water to 10,000 IU bottle of lyophilized HCG to create 10 stocks at 1,000 IU.
    Dilute each tube as needed with 20 ml embryo tested water to create 20 ml stocks at 50 IU/ml.
    Store at -20 °C for up to 6 weeks.
  3. Dilution of cytochalasin B
    Add 1 ml DMSO to 1 mg cytochalasin B powder to create 1 mg/ml stock solution.
    Dilute 5 μl stock in 1 ml M2 medium for a 5 μg/ml solution.
    Store at -20 °C for up to 6 months.
  4. Dilution of colcemid
    Add 10 μl of purchased, 10 μl/ml colcemid stock solution, and 1 ml M2 medium to create a 0.1 μg/ml solution.
    Store at -20 °C for up to 6 months.

Acknowledgments

This work was funded by the National Institutes for Health (grant numbers RO1 94518 and RO1103859 [to S.W.B.]), the National Foundation for Cancer Research (to D.R.W.), Susan G. Komen for the Cure (grant number SAC111370 [to D.R.W.]), U.S. Army Medical Research & Material Command (grant number W81XWH-07-1-0540d [to S.W.B.]) and a pilot grant from the University of Alabama at Birmingham Comprehensive Cancer Center (grant number CA013148 [to D.R.W. and S.W.B.]). Additional support was received from the National Institutes of Health (grant number RO1 HL109785 [to L.J.D.]), a National Institutes of Health predoctoral training programme in cardiovascular pathophysiology (grant number T32 HL007918 [to J.L.F.]), American Heart Association predoctoral fellowships (grant numbers 09PRE2240046 [to J.L.F.] and 11PRE7650033 [to K.J.D.]). The University of Alabama at Birmingham Transgenic Mouse Facility (RAK) is supported by the National Institutes of Health [grant numbers P30 CA13148, P30 AR048311, P30 DK074038, P30 DK05336 and P60 DK079626] and by the National Institutes of Health-funded Diabetes Research Center Bioanalytical Redox Biology Core [grant number P60 DK079626] located at the University of Alabama at Birmingham.

References

  1. Bayona-Bafaluy, M. P., Acin-Perez, R., Mullikin, J. C., Park, J. S., Moreno-Loshuertos, R., Hu, P., Perez-Martos, A., Fernandez-Silva, P., Bai, Y. and Enriquez, J. A. (2003). Revisiting the mouse mitochondrial DNA sequence. Nucleic Acids Res 31(18): 5349-5355.
  2. Betancourt, A. M., King, A. L., Fetterman, J. L., Millender-Swain, T., Finley, R. D., Oliva, C. R., Crowe, D. R., Ballinger, S. W. and Bailey, S. M. (2014). Mitochondrial-nuclear genome interactions in non-alcoholic fatty liver disease in mice. Biochem J 461(2): 223-232.
  3. Byers, S. L., Wiles, M. V., Dunn, S. L. and Taft, R. A. (2012). Mouse estrous cycle identification tool and images. PLoS One 7(4): e35538.
  4. Fetterman, J. L., Zelickson, B. R., Johnson, L. W., Moellering, D. R., Westbrook, D. G., Pompilius, M., Sammy, M. J., Johnson, M., Dunham-Snary, K. J., Cao, X., Bradley, W. E., Zhang, J., Wei, C. C., Chacko, B., Schurr, T. G., Kesterson, R. A., Dell'italia, L. J., Darley-Usmar, V. M., Welch, D. R. and Ballinger, S. W. (2013). Mitochondrial genetic background modulates bioenergetics and susceptibility to acute cardiac volume overload. Biochem J 455(2): 157-167.
  5. Freeman, H. C., Hugill, A., Dear, N. T., Ashcroft, F. M. and Cox, R. D. (2006). Deletion of nicotinamide nucleotide transhydrogenase: a new quantitive trait locus accounting for glucose intolerance in C57Bl/6J mice. Diabetes 55(7): 2153-2156.
  6. Grimsditch, D. C., Penfold, S., Latcham, J., Vidgeonhart, M., Groot, P. H. and Benson, G. M. (2000). C3H apoE(-/-) mice have less atherosclerosis than C57BL apoE(-/-) mice despite having a more atherogenic serum lipid profile. Atherosclerosis 151(2): 389-397.
  7. Han, Z., Cheng, Y., Liang, C. G. and Latham, K. E. (2010). Nuclear transfer in mouse oocytes and embryos. Methods Enzymol 476(2): 171-84.
  8. Kaku, K., Jr, F. F., Province, M. and Permutt, M. A. (1988).  Evidence for polygenic control. Diabetes 37(6): 707-13.
  9. Longenecker, G. and Kulkarni, A. B. (2009). Generation of gene knockout mice by ES cell microinjection. Curr Protoc Cell Chapter 19, Unit 19.14 19.14.1-36.
  10. Mckenzie, M., Trounce, I. A., Cassar, C. A., and Pinkert, C. A. (2004). Production of homoplasmic xenomitochondrial mice. Proc Natl Acad Sci U S A 101(6): 1685-1690.
  11. Paigen, B., Morrow, A., Brandon, C., Mitchell, D. and Holmes, P. (1990). Variation in susceptibility to atherosclerosis among inbred strains of mice. Atherosclerosis 10(2): 65-73.
  12. Wang, X., Ria, M., Kelmenson, P.M., Eriksson, P., Higgins, D.C., Samnegård, A., Petros, C., Rollins, J., Bennet, A.M., Wiman, B., de Faire, U., Wennberg, C., Olsson, P.G., Ishii, N., Sugamura, K., Hamsten, A., Forsman-Semb, K., Lagercrantz, J. and Paigen, B. (2005). Positional identification of TNFSF4, encoding OX40 ligand, as a gene that influences atherosclerosis susceptibility. Nat Genet 37(4): 365-372.
  13. Yu, X., Gimsa, U., Wester-Rosenlöf, L., Kanitz, E., Otten, W., Kunz, M. and Ibrahim S. M. (2009). Dissecting the effects of mtDNA variations on complex traits using mouse conplastic strains. Genome Res 19(1): 159-165.

简介

从植物组织提取的缩合单宁是酚醛树脂的合适替代物。它们的可能影响它们的化学反应性的分子结构可以通过在酸硫解和MALDI-TOF质谱之后使用HPLC-UV来评估。用半胱胺盐酸盐在酸性甲醇中溶解植物提取物导致缩合的单宁寡聚体的单体单元的释放,其可以通过与分析标准比较通过反相HPLC-UV进一步定量。使用2,5-二羟基苯甲酸作为基质和K sup +作为阳离子化试剂的MALDI-TOF质谱分析突出了单宁的分子结构特征(例如单体单元序列)低聚物。该方法允许估计平均和最大(可观察)聚合度,单体单元的类型和单宁单体的糖基化和/或酯化的存在。

[背景] 缩合单宁是由可从几种植物组织(例如软木树皮)中提取的黄烷-3-醇单体单元组成的多酚低聚物。它们已被认为是树脂配方(例如木材粘合剂和泡沫材料)中合成酚醛树脂的合适替代品。在缩合鞣酸中检测到的最常见的黄烷-3-醇单体,其羟基化模式和立体化学不同,如图1所示。

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图1.在缩合单宁结构中确定的最常见的单体

  低聚物中单体单元的具体结构和聚合度强烈影响单宁的化学反应性和物理性质,例如与醛的缩合反应速率,重金属螯合能力和水溶液的粘度溶液(Pizzi和Stephanou,1994; Yoneda和Nakatsubo,1998; Garnier等人,2001)。因此,鉴定单宁的分子结构对于更好地确定其可能的利用是重要的。
  缩合单宁单体的结构的分析已经用不同的方法进行,例如尺寸排阻色谱法(SEC),正相和反相高效液相色谱法(HPLC),基质辅助激光解吸离子化飞行时间质谱(MALDI-TOF MS)和核磁共振(NMR)(Hümmer和Schreier,2008)。
 在酸性甲醇中和在甲苯-α-硫醇或半胱胺盐酸盐(硫解)的存在下,然后通过反相HPLC-UV的单宁的解聚被认为是用于估计缩合单宁的平均聚合度的合适方法,它们的构建单元的构型(Matthews等人,1997; Cheynier等人,2001; Jerez等人, ,2007; Bianchi等人,2015)。同样,已经显示MALDI-TOF MS在确定缩合单宁的结构方面是成功的(Pasch等人,2001; Monagas等人,2010 ; Bianchi 等。,2014)。
   用于分析从植物组织提取的缩合单宁的所提出的方法代表了先前公布的酸性硫解和MALDI-TOF MS之后的HPLC-UV程序的优化。进行HPLC分析中流动相梯度的仔细调节,以便在释放的黄烷醇和它们相应的硫醚之间达到充分的分离。在制备用于MALDI-TOF MS的样品中的阳离子化试剂的选择也被仔细地测量,特别是考虑到含有相对高量的无机化合物如树皮提取物的样品。
 该方法成功地用于表征从软木树种,例如银杉,欧洲落叶松,挪威云杉,道格拉斯杉木和苏格兰松树提取的缩合单宁(Bianchi等人。,2015)。...

关键字:线粒体, 线粒体DNA, 多态性, 串扰

材料和试剂

  1. 26号针
  2. 1或3毫升注射器
  3. 显微镜载玻片
  4. CellTram Vario注射器
  5. 10厘米组织培养皿
  6. 所需的核或线粒体遗传背景的女性供体小鼠(3-4周龄)
  7. 作为捐赠者女性的匹配核背景的雄性育种者(经证明)
  8. 女性受体小鼠(8-10周龄)
  9. 切除的雄性小鼠(经证实)
  10. 来自怀孕母马血清(PMS)(Sigma-Aldrich,目录号:G4877)的促性腺激素
    注意:此产品已停产。
  11. 人绒毛膜促性腺激素(HCG)(Sigma-Aldrich,目录号:CG10)
  12. M2培养基(Sigma-Aldrich,目录号:M7167)
  13. 来自Drechslera dematioidea的细胞松弛素B(Sigma-Aldrich,目录号:C6762)
  14. Colcmid(Sigma-Aldrich,目录号:D1925)
  15. 胚胎测试的矿物油(Sigma-Aldrich,目录号:M8410)
  16. 胚胎移植用水(Sigma-Aldrich,目录号:W1503)
  17. 限制酶:
    1. BclI(New England Biolabs)
    2. PflF1/AspI(New England Biolabs)
  18. 稀释PMS(参见配方)
  19. HCG的稀释(参见配方)
  20. 细胞松弛素B的稀释(参见配方)
  21. 秋水仙的稀释(见配方)

设备

  1. 压电钻(压电钻)(Sutter仪器,型号:PrimeTech PMM-150FU)
  2. 电穿孔仪(BTX The Electroporation Experts,型号:ECM 630)
  3. 微量移液器(水平移液管拉出器)(Sutter Instrument,型号:P-87)
    产品P-87已停产,可用的产品是P-97。
  4. Micropipette微型铸造(Defonbrune微型铸造显微镜头)(Leitz)
  5. 保持移液器拉出器(垂直移液器拉出器)(David Kopf Instrument,型号:720)
  6. 显微镜(Laborlux S Nomarski DIC)(Leica Microsystems)
  7. CellTram Vario注射器(Eppendorf)
  8. 台式孵化器(Cook,型号:MINC G20079)
  9. 45度角钳(Fine Science Tools,目录号:11251-35)
  10. 直镊子(Fine Science Tools,目录号:11251-10)
  11. 微切割弹簧剪(Roboz Surgical Instrument,目录号:RS-5650)
  12. 嘴移液器

程序

  1. 创建MNX小鼠


    图1.整个程序示意图。通过从"菌株A"(C57BL/6)和"菌株B"(C3H/HeN)小鼠摘除受精卵细胞产生MNX小鼠。 MNX命名由菌株核(Strain )表示:菌株mtDNA(Strain ),例如。具有C3H核DNA和C57的小鼠mtDNA由C3H支持物表示:C57 mt 。将菌株B原核转移至去核的菌株A卵母细胞,产生菌株B:菌株A (C 3 H :C57 < )卵母细胞,将其植入替代雌性以产生MNX后代。遵循相互过程以产生菌株A :菌株B (C57 :C3H mt ):卵母细胞
    1. 使用26号针头和1或3ml注射器,用PMS(5.0-7.5IU,体积0.2-0.3ml)腹膜内(IP)注射雌性供体小鼠。小鼠的最佳剂量和年龄可以是菌株特异性的,并且应当通过剂量响应曲线和受精胚胎产生的评估来确定。通常,雌性小鼠在第1天中午注射。
    2. 在(第3天)PMS注射后约48小时,供体小鼠接受与PMS相同剂量的IPG注射的HCG。在注射后,超排卵的雌性与配对并且与具有相同核基因型(与女性相匹配,图1)的已证明的育种雄性配对;经常交配发生在交配的前1-2小时内。通过目视检查呈阳性征象的雌性受体小鼠与证实的切除血管的雄性配对以产生假孕(Byers等人,2012)。
    3. 第二天早上检查阴道栓塞的女性。插塞的存在表示成功的交配,但不一定表示受精。然而,没有插头不能确认没有发生成功的育种,因为插头的容易识别可能是应变依赖的
    4. 如前所述收获菌群以产生单细胞胚(图2A和2B)(Han等人,2010)。


      图2.从输卵管收集卵丘块。用阴道塞从超量配置的雌性解剖输卵管(A)。肿胀的壶腹(由箭头指示)切口以允许排出胚胎和卵丘细胞(B)。

    5. 通过嘴移液管将原核胚胎置于含有细胞松弛素B(5μg/ml)和秋水仙碱(0.1μg/ml)的M2培养基中,在37℃下放置5分钟,并保持在显微镜载片上的M2培养基的显微注射液滴温度以防止操作过程中的溶解
    6. 使用与用于胚胎干细胞注射(大约20微米内径)的斜角移液管相似的大小和形状的微量移液管通过在第一胚胎的透明带上施加轻微压力来除去每个胚胎的原核(Longenecker和Kulkarni, 2009)。高强度压电脉冲仅施加直到zona破裂(大约1-2秒),然后关闭。将移液管缓慢推进至每个原核(图3),并且使用CellTram vario注射器将温和的抽吸施加至针,将两个原核作为单个单元(karyoplast,图3)吸出并移出。只有当原核与针接触时,适当的针尺寸和吸力的应用最小化了细胞质内容物进入针的吸收。
    7. 在相互胚胎上重复该过程,导致移液管含有来自两种菌株的原核(图3)。


      图3.前核的分离(A)将移液管缓慢推进到每个原核(由黑色箭头指示),并使用CellTram vario注射器(B)将原核作为单个单元(karyoplast,K)吸出并移出(C)。 ZP,透明带
    8. 将来自菌株A的分离的原核植入菌株B的去核胚胎中(图4)。将来自菌株B的相互的核质体置于去核的菌株A胚胎中

      图4.原核转移然后将来自一个菌株的核质体(K)植入到互逆菌株的去核胚胎中。 ZP,透明带。 (A)插入包含核质体的移液管(B)核质体的排出(C)重建的胚胎。

    9. 十厘米组织培养皿(图5)装载有30μl的M2培养基的微滴并覆盖在矿物油中,并且通过嘴移液管将每个胚胎放入其自身的培养基滴中。将菌株A和菌株B胚胎置于分开的皿中。将电极放置在将两个极之间的胚胎定位的液滴中。对每个重构的合子施加单个90V脉冲,并将所有受精卵培养过夜(Han等人,2010)。


      图5.胚胎培养皿。图1-8是含有重构的合子的不同的M2培养基和矿物油滴。每一滴可以含有1-2个受精卵
    10. 将两个细胞的胚胎转移到0.5天假孕小鼠的输卵管口上;每个受体转移大约20个胚胎(图6)

      图6.重建胚胎的输卵管转移。通过口腔吸管将两个细胞胚胎通过漏斗口放入假孕受体小鼠的输卵管中。

    11. 核基因型和mtDNA单倍型在后代通过核SNP和完整的mtDNA基因组序列分析从耳尾或尾夹验证。

  2. MNX小鼠集落建立
    基于对每种潜在mtDNA单倍型特异性的PCR分析,使用同质性创始者雌性MNX小鼠建立MNX小鼠集落。最初通过对每种单倍型开发的PCR检测方法和通过测序确认的同质雌性确定mtDNA单倍型。期望的同质(基于PCR筛选)在约20%-30%的产生的雌性中鉴定用作创始者的雌性。
    1. MNX女性的验证mtDNA序列和表观同质性(使用耳朵或尾夹DNA)与匹配核背景的男性交叉以产生F1窝。
    2. 通过对诊断mtDNA SNP特异性设计的PCR分析,测试来自F1后代的多个组织以验证期望的mtDNA单倍型/同形型(Bayona-Bafaluy等人,2003)。例如,将F1 C57阳性和C3H阳性:C57 小鼠在基于多种组织的基因组中进行单倍群分组在PCR检测方法中询问异质性的潜力(图7)。
      PCR方案:
      2x GoTaq MasterMix:25μl
      引物:每个5μl
      无核酸酶H 2 O:12.5μl
      DNA:2.5μl
      热循环特性:
      95℃2分钟:1个循环
      95℃30秒; 57℃1分钟; 72℃30秒:35个循环
      72℃10分钟:1个循环
      24°C无穷
      1. 筛选bp 9461(204碱基对扩增子)处的ND3突变:
        9461F:5'-TTCCAATTAGTAGATTCTGAATAAACCCAGAAGAGAGTGAT-3'
        9461R:5'-AAATTTTATTGAGAATGGTAGACG-3'
        在10微升反应体积中用限制酶Bcli(10U,New England Biolabs)消化10微升扩增子。 C3H mtDNA被切割成166bp和38bp片段,而C57mtDNA保持未切割。
      2. 筛选bp 9348 CO3突变(385碱基对扩增子):
        9348F:5'-CGAAACCACATAAATCAAGCCC-3'
        9348R:5'-CTCTCTTCTGGGTTTATTCAGA-3'
        使用Pflf1(AspI,10U,New England Biolabs)限制酶在20微升反应体积中消化10微升扩增子。 C3H mtDNA保持未切割,而C57 mtDNA被消化成274bp和111bp片段。


        图7.线粒体 - 核交换(MNX)小鼠的生成。所有起始创始雌性和F1后代的核基因分型和mtDNA单元型通过一组38个区分核SNP的核SNP分析和完整的mtDNA测序。通过对创始人的直接测序进行初始mtDNA单倍型验证后,通过使用AspI和BclI的限制性酶长度多态性分析对后续世代进行单倍型分析,其给出C3H和C57mtDNA的不同模式。来自C57mtDNA的PCR产物保持未切割(204bp),而C3H mtDNA被BclI切割产生166bp和38bp片段。 B.来自C57 mtDNA的PCR产物被AspI切割产生274bp和111bp片段,而C3H mtDNAs保持未切割(385bp)。

    3. 产生在所有测试组织中出现同质的小鼠的MNX雌性创建者然后用作每个相应MNX集落的建立水坝。
    4. 使用基于PCR的技术,每一代的所有后代是mtDNA单倍群,以验证期望的遗传背景的维持。

数据分析

详细描述MNX小鼠的产生以及分析和复制信息的原始研究文献可在网上获得(Fetterman等人,2013)。

食谱

  1. 稀释PMS
    将20毫升胚胎测试水加入到1000 IU瓶的冻干PMS中,以50 IU/ml产生20毫升原液。
    储存于-20°C长达3个月。
  2. 稀释HCG
    加入10ml胚胎测试水至10,000IU瓶的冻干HCG以在1,000IU产生10个储备物。
    根据需要用20ml胚胎测试水稀释每个试管,以50IU/ml产生20ml原液 -20℃保存6周。
  3. 细胞松弛素B的稀释
    加入1ml DMSO至1mg细胞松弛素B粉末,以产生1mg/ml储备溶液 稀释5微升储备在1毫升M2介质为5微克/毫升的解决方案。
    储存于-20°C长达6个月。
  4. 阴离子稀释
    加入10μl购买的,10μl/ml秋水仙素储备溶液和1ml M2培养基以产生0.1μg/ml溶液。
    储存于-20°C长达6个月。

致谢

这项工作由国家卫生研究院资助(授权号RO1 94518和RO1103859 [到SWB]),国家癌症研究基金会(DRW),Susan G. Komen治疗(授权号SAC111370 [到DRW]) ,US Army Medical Research&材料指令(授权号W81XWH-07-1-0540d [至S.W.B.])和来自阿拉巴马大学在伯明翰综合癌症中心的试验性授权(授权号CA013148 [授予D.R.W.和S.W.B.])。从美国国立卫生研究院(授予号RO1 HL109785 [到LJD]),国家卫生研究院的心血管病理生理学(授予号T32 HL007918 [到JLF]),美国心脏协会退变性奖学金(授予号)数字09PRE2240046 [to JLF]和11PRE7650033 [to KJD])。阿拉巴马大学在伯明翰转基因小鼠设备(RAK)由国立卫生研究院[资助号P30CA13148,P30AR048311,P30DK074038,P30DK05336和P60DK079626]支持,以及由国立卫生研究院资助的糖尿病研究中心位于伯明翰阿拉巴马大学的生物分析氧化还原生物学核心[授予号P60 DK079626]。

参考文献

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引用:Kesterson, R. A., Johnson, L. W., Lambert, L. J., Vivian, J. L., Welch, D. R. and Ballinger, S. W. (2016). Generation of Mitochondrial-nuclear eXchange Mice via Pronuclear Transfer. Bio-protocol 6(20): e1976. DOI: 10.21769/BioProtoc.1976.
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