DNA显微注射法生成秀丽隐杆线虫转基因动物

Matthias  Rieckher; Nektarios  Tavernarakis

doi:10.21769/BioProtoc.2565

Improve Research Reproducibility A Bio-protocol resource

提交稿件
订阅
登录
/
注册
- 个人主页
- 编辑个人信息
- 修改密码
- 退出
CN
- EN - English
- CN - 中文

Peer-reviewed

Generation of Caenorhabditis elegans Transgenic Animals by DNA Microinjection

DNA显微注射法生成秀丽隐杆线虫转基因动物

Matthias Rieckher

Nektarios Tavernarakis email

发布: 2017年10月05日第7卷第19期 DOI: 10.21769/BioProtoc.2565 浏览次数: 14836

评审: Jyotiska ChaudhuriManish ChamoliAnonymous reviewer(s)

PDF

Q&A

引用

Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Methods in Molecular Biology, featuring study using the protocol.

May 2009

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

相关实验方案

聚乙二醇介导的圆锥掘氏梅里霉的转化

Le D. He and Jonathan J. Ewbank

2017年03月05日 9001 阅读

非模式微生物桃色欧文氏菌的高效遗传转化及自杀质粒介导的基因组编辑系统

Tingfeng Cheng [...] Lei Zhao

2024年03月20日 1505 阅读

家蚕卵的显微注射操作方法

Hayato Yamada [...] Teruyuki Niimi

2025年05月20日 2204 阅读

Abstract

Microinjection is the most frequently used tool for genetic transformation of the nematode Caenorhabditis elegans, facilitating the transgenic expression of genes, genome editing by the clustered regularly interspersed short palindromic repeats (CRISPR)-Cas9 system, or transcription of dsRNA for RNA intereference (RNAi). Exogenous DNA is delivered into the developing oocytes in the germline of adult hermaphrodites, which then generate transgenic animals among their offspring. In this protocol, we describe the microinjection procedure and the subsequent selection of transgenic progeny.

Keywords: Caenorhabditis elegans (秀丽隐杆线虫)

Genetic transformation (遗传转化)

Microinjection (显微注射)

Extrachromosomal arrays (染色体外阵列)

Germline (生殖细胞系)

GFP (GFP)

Transgenic animals (转基因动物)

Background

In C. elegans, DNA transformation by microinjection is commonly used to produce transgenic animals that over-express or ectopically express genes, which can be fused to tags (e.g., green fluorescent protein [GFP]), allowing for the phenotypic rescue of mutants, and/or the analysis of localization and function of proteins (Carter et al., 1990; Chalfie et al., 1994; Mello and Fire, 1995). The advent of the clustered regularly interspersed short palindromic repeats (CRISPR)-Cas9 system requires microinjection to achieve highly specific genome editing by introducing point mutations or insertion/deletion mutations (summarized in Dickinson and Goldstein, 2016). Further, the technique is applied for the inducible and/or tissue-specific in vivo transcription of dsRNA to facilitate heritable RNA interference (RNAi) (Tavernarakis et al., 2000). The genetic material is injected into the distal gonad syncytium of adult hermaphrodite animals where it enters the developing oocytes (Figure 1). The exogenous DNA is arranged into large extrachromosomal arrays consisting of 50 to 300 copies, which are inherited in a non-Mendelian fashion to the following generations. Next-generation progeny (F1) carry the extrachromosomal array with a varying probability (between 5 to 80% [Stinchcomb et al., 1985; Mello et al., 1991]). Transgenic animals are identified amongst the offspring by the use of co-transformation markers, which are injected along with the DNA of interest (Table 1). Most commonly used are the pharyngeal expression of green fluorescent protein (GFP) or red fluorescence (mCherry), or the dominant rol-6(su1006) allele, which induces a distinct rolling phenotype in transgenic F1 progeny (Mello et al., 1991; Tabara et al., 1996; Frokjaer-Jensen et al., 2008).

Table 1. Commonly used co-transformation markers with distinct phenotypes for selection of transgenic animals upon microinjection in C. elegans

Figure 1. Microinjection scheme for C. elegans. A. Scheme of an adult C. elegans displaying the major organs including the pharynx, intestine and the gonad. When microinjecting C. elegans the injection capillary needs to be inserted in the syncytium (cytoplasmic core) of the distal gonad. The inlay indicates the area of interest for injection. B. DIC image of the area indicated in 1A. The nuclei of the germ-cells are clearly visible and surround the syncytium. Size bar corresponds to 50 µm.

Materials and Reagents

Sterile pipette tips
Microloader pipette tips (Eppendorf, catalog number: 5242956003 )
Microinjection capillaries (Eppendorf Femtotips II, 0.5 µm inner and 0.7 µm outer diameter) (Eppendorf, catalog number: 930000043 )
Microscope slides, 76 x 26 mm (Carl Roth, catalog number: 0656.1 )
Microscope cover glasses, 24 x 60 mm (VWR, catalog number: 631-1575 )
Tape (~1 mm thickness)
Greiner Petri dishes (60 x 15 mm) (Sigma-Aldrich, catalog number: P5237 )
Glass Pasteur Pipettes, disposable, 150 mm (BRAND, catalog number: 747715 )
C. elegans animals (e.g., available from Caenorhabditis Genetics Center [CGC], University of Minnesota, MN, USA)
Escherichia coli OP50 strain (obtained from the Caenorhabditis Genetics Center)
DNA of interest for injection (plasmid, fosmid, PCR fragment, or similar) and plasmid containing a co-transformation marker (see Table 1)
Miniprep Kit for DNA purification (e.g., QIAGEN-tip 20) (QIAGEN, catalog number: 10023 )
Halocarbon oil 700 (Sigma-Aldrich, catalog number: H8898-100ML )
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888 )
Peptone (BD, Bacto^TM, catalog number: 211677 )
Streptomycin sulfate salt (Sigma-Aldrich, catalog number: S6501 )
Agar (Sigma-Aldrich, catalog number: 05040 )
100% ethanol
Calcium chloride dehydrate (CaCl₂·2H₂O) (Sigma-Aldrich, catalog number: C5080 )
Magnesium sulfate (MgSO₄) (Sigma-Aldrich, catalog number: M7506 )
Cholesterol stock solution (SERVA Electrophoresis, catalog number: 17101.01 )
Nystatin stock solution (Sigma-Aldrich, catalog number: N3503 )
Potassium dihydrogen phosphate (KH₂PO₄) (Carl Roth, catalog number: P018.1 )
Di-Sodium hydrogen phosphate (Na₂HPO₄) (Carl Roth, catalog number: T876.1 )
Di-Potassium hydrogen phosphate (K₂HPO₄) (Carl Roth, catalog number: 5066.1 )
Agarose (Biozym Scientific, catalog number: 840004 )
OP50 seeded NGM agar plates (see Recipes)
2% agarose pads (see Recipes)
Hairpin pick (see Recipes)
M9 buffer (see Recipes)

Equipment

Bunsen burner
Cylindrical glass beaker
Wormpick with platinum wire and pre-flattened tip (Genesee Scientific, catalog numbers: 59-AWP and 59-30P6 )
Microwave
Heat plate (e.g., IKA, model: C-MAG HP 4 , catalog number: 0003581600)
Puller (optional; e.g., Sutter Instrument, model: P-97 )
Autoclave
Stereomicroscope (e.g., Leica Microsystems, model: Leica M80 ), optionally fluorescent stereomicroscope (e.g., Leica Microsystems, model: Leica M165 FC)
Microscope for Microinjection (ZEISS, model: Axio Observer.A1 equipped with differential interference contrast [DIC] prisms, gliding table, objective 10x/0.3 M27, objective 40x/0.75 M27, optionally camera AxioCam ERc 5 sec and monitor for observation and demonstration purposes)
Micromanipulator (e.g., Eppendorf InjectMan 4) (Eppendorf, catalog number: 5192000019 )
Microinjector unit (e.g., Eppendorf FemtoJet 4i) (Eppendorf, catalog number: 5252000013 )
Microcentrifuge (e.g., Eppendorf Centrifuge 5424) (Eppendorf, model: 5424 , catalog number: 5404000014)
Spectrophotometer (e.g., Thermo Fisher Scientific, model: NanoDrop^TM 8000 , catalog number: ND-8000-GL)

Procedure

English

中文翻译

文章信息

版权信息

如何引用

Rieckher, M. and Tavernarakis, N. (2017). Generation of Caenorhabditis elegans Transgenic Animals by DNA Microinjection. Bio-protocol 7(19): e2565. DOI: 10.21769/BioProtoc.2565.