在优化微环境中利用杆状病毒和质粒载体介导的大型蚤胚胎原代细胞基因转导：方法与方案

C.P. Sreevidya; Soumya Balakrishnan; Jayesh Puthumana

doi:10.21769/BioProtoc.5266

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Baculovirus and Plasmid Vector-Mediated Gene Transfer in Daphnia magna Cells in Primary Embryonic In Vitro Cultures in an Optimized Microenvironment: Methods and Protocols

在优化微环境中利用杆状病毒和质粒载体介导的大型蚤胚胎原代细胞基因转导：方法与方案

CS C.P. Sreevidya

SB Soumya Balakrishnan

JP Jayesh Puthumana email

发布: 2025年04月05日第15卷第7期 DOI: 10.21769/BioProtoc.5266 浏览次数: 385

评审: Alberto RissoneSrestha DasguptaKeisuke Tabata

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Abstract

Daphnia magna is a well-established model organism in ecotoxicology, environmental monitoring, and genetics due to its sensitivity to pollutants, its pivotal role in freshwater ecosystems, and its well-characterized genome. Despite its extensive use in these fields, there is a notable lack of established protocols for developing primary cell culture systems and conducting transgenic studies in Daphnia spp. This study addresses these gaps by optimizing a medium and standardizing protocols for primary cell culture and transgenic experiments in D. magna. Primary cell cultures were established from both D. magna embryos and whole organisms, with medium optimization verified using XTT assay. Cell viability was sustained for over two months using a modified Schneider’s insect medium enriched with FBS, glucose, MEM vitamin mix, and selenium. DNA replication and cell proliferation were confirmed through BrdU labeling. Both mechanical and enzymatic passaging methods were compared, resulting in 20% and 10% cell attachment, respectively. For transgenic applications, this study successfully standardized plasmid-mediated lipofection and baculovirus-mediated transduction, achieving success rates of 52% and 45%. These findings represent a pioneering effort in D. magna embryonic cell culture, offering a reliable in vitro platform for future biological research, including ecotoxicological and epigenetic investigations. The established protocols and optimized cell culture medium have significant implications for advancing crustacean cell line research and transgenic model development, enhancing our understanding of biological processes in controlled laboratory environments.

Key features

• Optimized primary embryonic cell culture system for Daphnia magna ensures extended viability and minimized contamination compared to adult-derived cultures.

• Modified Schneider’s insect medium (MSIM) with precise supplement concentrations enhances Daphnia cell viability, supporting applications in toxicity testing and genetic modifications.

• Efficient transfection and baculovirus-based transduction protocols enable robust transgene expression, validated through fluorescence microscopy and promoter-specific activity.

• Comparative subculture techniques demonstrate improved reattachment rates with gentle pipetting, providing insights into non-enzymatic handling for fragile aquatic cells.

Keywords: Daphnia magna (大型蚤)

Primary cell culture (原代细胞培养)

Optimized microenvironment (优化微环境)

Transfection (转染)

Transduction (转导)

Recombinant baculovirus expression system (重组杆状病毒表达系统)

Graphical overview

Primary cell culture development from Daphnia magna embryos

Background

Crustaceans, encompassing ~67,000 species, include Daphnia, a keystone species in freshwater ecosystems known for their adaptability, parthenogenetic reproduction, and utility in evolutionary and ecotoxicological studies. Despite Daphnia’s potential as a model organism, the lack of established cell lines limits in vitro research and pathogen studies [1]. Challenges in crustacean cell culture primarily stem from the absence of suitable media [2,3], which has prompted modifications of commercially available media to optimize cell viability and microenvironments.

Previous attempts to develop crustacean cell lines have faced poor cell survival [1–3]. Notable successes include hybrid lines like PmLyO-Sf9 [4], but efforts for Daphnia magna have yielded limited results [5,6], emphasizing the need for optimized protocols. Advances in Daphnia genomic tools and transgenic methods, including RNAi [7], targeted mutagenesis [8], and genome editing [9], have paved the way for genetic manipulation. Cellfectin has shown higher efficiency than lipofectamine in Arthropoda [10]. The EF1α-DsRed plasmid, modified as pRCS21-EF1α1(UTR), achieved 0.67% transgenic D. magna via microinjection [11]. Baculovirus expression systems, mainly Autographa californica MNPV, enhance protein expression using polyhedrin or p10 promoters. However, challenges persist in transfection and transduction due to low cell numbers and attachment efficiency in novel cultures.

This study establishes a standardized protocol for developing primary D. magna embryonic cell cultures using a modified medium to enhance cell survival. Transfection and transduction protocols are optimized to create an immortalized D. magna cell line, offering broad applications in crustacean research and biotechnology.

Materials and reagents

Biological materials

1. Daphnia magna: Daphnia Research Facility (DRF), National Centre for Aquatic Animal Health (NCAAH; https://www.ncaah.ac.in), Cochin University of Science and Technology (CUSAT; https://cusat.ac.in/), Kerala, India

2. pCS-EF1α1-DSRed2 vector, obtained from Dr. Hajime Watanabe, Osaka University, Japan [10]

3. Recombinant baculovirus expression system (BacIe1-GFP and BacP2-GFP), developed in NCAAH, CUSAT [2,3]

4. SF9 cells, maintained at NCAAH, CUSAT

Reagents

1. MEM vitamins (MEM) (Gibco, catalog number: 11120052)

2. 1× PBS (Gibco, catalog number: 10010023)

3. Schneider’s insect medium (Gibco, catalog number: 21720024)

4. Fetal bovine serum (FBS) (Gibco, catalog number: A5670701)

5. 1× Leibovitz’s L-15 medium (L-15) (Sigma-Aldrich, catalog number: L1518)

6. TNM-FH insect medium (TNMFH) (Sigma-Aldrich, catalog number: T1032)

7. Grace’s insect medium (GIM) (Sigma-Aldrich, catalog number: G9771)

8. Minimal Essential Medium (MEM) (Sigma-Aldrich, catalog number: 56416C)

9. Dulbecco’s modified Eagle medium (DMEM) (Sigma-Aldrich, catalog number: D6546)

10. Roswell Park Memorial Institute Medium (RPMI) (Sigma-Aldrich, catalog number: R8758)

11. XTT (Invitrogen, catalog number: X6493)

12. Glucose, ≥99.5% (Sigma-Aldrich, catalog number: G7021)

13. Trehalose, ≥99% (Sigma-Aldrich, catalog number: T0167)

14. Sucrose, ≥99% (Sigma-Aldrich, catalog number: S5390)

15. Amino acids mix (MEM) (Gibco, catalog number: 11130036)

16. Cholesterol (Sigma-Aldrich, catalog number: C4951)

17. Sodium selenite, ≥98% (Sigma-Aldrich, catalog number: S5261)

18. Trypsin-EDTA solution (Sigma-Aldrich, catalog number: T4049)

19. Acridine orange (AO) (Invitrogen, catalog number: A1301)

20. Ethidium bromide (EB) (Invitrogen, catalog number: 15585011)

21. BrdU (5-Bromo-2’-deoxyuridine) (TCI, catalog number: B1575)

22. Mouse monoclonal anti-BrdU antibody (Sigma-Aldrich, catalog number: SAB4700630)

23. Alexa Fluor 488 (Jackson ImmunoResearch Labs, catalog number: 115-545-003)

24. DAPI (Thermo Fisher Scientific, catalog number: D3571)

25. Cellfectin (Cellfectin^TM II) (Invitrogen, catalog number: 10362100)

26. Selenium dioxide (SeO₂) (Sigma-Aldrich, catalog number: 204315)

27. Penicillin (100 IU/mL) and streptomycin (100 μg/mL) solution (Roche, Merk, catalog number: 11074440001)

Solutions

1. ADaM media (see Recipes)

2. 0.05% sodium hypochlorite (see Recipes)

3. 70% ethanol (see Recipes)

4. 2 M HCl (see Recipes)

5. 0.1 M sodium borate (see Recipes)

6. 0.2% Triton X-100 (see Recipes)

7. 3% BSA (see Recipes)

8. 0.05% trypsin-EDTA (see Recipes)

Recipes

1. ADaM media

Reagent	Final concentration	Quantity or Volume
CaCl₂·2H₂O	0.2705 g/L	23 mL of Stock A (117.6 g/L CaCl₂·2H₂O)
NaHCO₃	0.0554 g/L	22 mL of Stock B (25.2 g/L NaHCO₃)
SeO₂	7 μg/L	1 mL of Stock C (0.07 g/L SeO₂)
Distilled water		Up to 10 L

2. 0.05% sodium hypochlorite

Reagent	Final concentration	Quantity or Volume
Sodium hypochlorite powder	0.05% (w/v)	0.05 g (for 10 mL)
Distilled water	n/a	Up to 10 mL
Total	n/a	10 mL

3. 70% ethanol

Reagent	Final concentration	Quantity or Volume
Ethanol (100%)	70% (v/v)	7 mL
Distilled water	n/a	3 mL
Total	n/a	10 mL

4. 2 M HCl

Reagent	Final concentration	Quantity or Volume
HCl (12 M)	2 M	1.67 mL
Distilled water	n/a	8.33 mL
Total	n/a	10 mL

5. Sodium borate (0.1 M)

Reagent	Final concentration	Quantity or Volume
Sodium borate	0.1 M	0.382 g
Distilled water	n/a	9.618 mL
Total	n/a	10 mL

6. 0.2% Triton X-100

Reagent	Final concentration	Quantity or Volume
Triton X-100	0.2%	20 mg
Distilled water	n/a	10 mL
Total	n/a	10 mL

7. 3% BSA

Reagent	Final concentration	Quantity or Volume
3% BSA	3%	0.3 g
Distilled water	n/a	10 mL
Total	n/a	10 mL

8. 0.05% trypsin-EDTA

Reagent	Final concentration	Quantity or Volume
Trypsin-EDTA stock	0.25%	2 mL
Distilled water	n/a	8 mL
Total	n/a	10 mL

Laboratory supplies

1. 1.5 mL microcentrifuge tubes (MCTs) (Tarson, catalog number: 500010)

2. 2 mL microcentrifuge tubes (MCTs) (Tarson, catalog number: 500020)

3. Needles (Sigma-Aldrich, catalog number: Z117013)

4. Glass homogenizer (Sigma-Aldrich, catalog number: D8938)

5. 96-well plate (TPP, catalog number: Z707902)

6. 24-well plate (TPP, catalog number: Z707791)

7. Coverslips (Sigma-Aldrich, catalog number: Z355445)

8. Pipette tips: 1–10, 10–100, 100–1,000 μL (Sigma-Aldrich, catalog numbers: Z678295, Z678325, AXYT1000CR)

Equipment

1. Fluorescent microscope (Invitrogen, model: AMF7000, EVOS^TM M7000 Imaging System)

2. Centrifuge (Eppendorf, model: 5424R, catalog number: 5404000138)

3. Dark incubator, 28 °C (Thermo Fisher Scientific, catalog number: 3120, model: Forma Series II)

4. Laminar airflow (Thermo Scientific, catalog number: 51025413, MSC-Advantage^TM Class II Biological Safety Cabinets)

Software and datasets

1. SPSS^® software (version 21; SPSS Inc., Chicago, IL, USA)

Procedure

English

中文翻译

文章信息

稿件历史记录

提交日期: Dec 25, 2024

接收日期: Mar 2, 2025

在线发布日期: Mar 24, 2025

出版日期: Apr 5, 2025

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如何引用

Sreevidya, C. P., Balakrishnan, S. and Puthumana, J. (2025). Baculovirus and Plasmid Vector-Mediated Gene Transfer in Daphnia magna Cells in Primary Embryonic In Vitro Cultures in an Optimized Microenvironment: Methods and Protocols. Bio-protocol 15(7): e5266. DOI: 10.21769/BioProtoc.5266.