通过原核移植生成线粒体-细胞核置换小鼠

Robert A.  Kesterson; Larry W. Johnson; Laura J. Lambert; Jay L. Vivian; Danny R. Welch; Scott W. Ballinger

doi:10.21769/BioProtoc.1976

Improve Research Reproducibility A Bio-protocol resource

提交稿件
订阅
CN
- EN - English
- CN - 中文

Peer-reviewed

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

Robert A. Kesterson email

LJ Larry W. Johnson

LL Laura J. Lambert

JV Jay L. Vivian

DW Danny R. Welch email

Scott W. Ballinger email

发布: 2016年10月20日第6卷第20期 DOI: 10.21769/BioProtoc.1976 浏览次数: 11468

评审: Masahiro MoritaAnonymous reviewer(s)

下载 PDF

Q&A

引用

Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Cancer Research, featuring study using the protocol.

Oct 2015

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

相关实验方案

条件性人 BRD4 敲入转基因小鼠基因分型和蛋白质异构体检测

Michael Paul Lewis [...] Cheng-Ming Chiang

2022年04月05日 2491 阅读

用于癌症遗传依赖性功能验证的阵列式 CRISPR/Cas9 筛选

Ludovica Proietti [...] Florian Grebien

2022年12月20日 2299 阅读

利用环状聚合酶扩展克隆（CPEC）方法构建CRISPR筛选文库

Bengisu Dayanc [...] Serif Senturk

2025年02月20日 1457 阅读

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

26 gauge needle
1 or 3 ml syringe
Microscope slides
CellTram vario syringe
10 centimeter tissue culture dishes
Female donor mice (3-4 weeks of age) of desired nuclear or mitochondrial genetic backgrounds
Male breeders (proven) of matching nuclear background as donor females
Female recipient mice (8-10 weeks of age)
Vasectomized male mice (proven)
Gonadotropin from pregnant mare serum (PMS) (Sigma-Aldrich, catalog number: G4877 )
Note: This product has been discontinued.
Human chorionic gonadotropin (HCG) (Sigma-Aldrich, catalog number: CG10 )
M2 medium (Sigma-Aldrich, catalog number: M7167 )
Cytochalasin B from Drechslera dematioidea (Sigma-Aldrich, catalog number: C6762 )
Colcemid (Sigma-Aldrich, catalog number: D1925 )
Embryo tested mineral oil (Sigma-Aldrich, catalog number: M8410 )
Water for embryo transfer (Sigma-Aldrich, catalog number: W1503 )
Restriction enzyme:

BclI (New England Biolabs)
PflFI/AspI (New England Biolabs)

Dilution of PMS (see Recipes)
Dilution of HCG (see Recipes)
Dilution of cytochalasin B (see Recipes)
Dilution of colcemid (see Recipes)

Equipment

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

Procedure

English

中文翻译

文章信息

版权信息

如何引用

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.