Establishment and Optimization of an Electroporation-Mediated Transfection System in Dwarf Surf Clam Mulinia lateralis
SUN Xiaohan1,2, DAI Shukun1,2, LIN Siyu1,2, GUO Xiaolin1,2, ZHAO Kaixuan1,2, YANG Zujing1,2, ZHANG Zhifeng1,2,3, QIN Zhenkui1,2
1. Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao 266003, China; 2. Shandong Key Laboratory of Marine Seed Industry (preparatory), Ocean University of China, Qingdao 266003, China; 3. Key Laboratory of Tropical Aquatic Germplasm of Hainan Province, Sanya Oceanographic Institution, Ocean University of China, Sanya 572000, China
Abstract：Exogenous gene introduction technology plays a pivotal role in trait improvement of economically important aquatic species, particularly shellfish. However, the limitations of related technological platforms severely restrict the elucidation of molecular mechanisms underlying critical traits and the advancement of genetic breeding. To identify suitable exogenous gene delivery methods for application in shellfish, a fluorescent reporter gene vector was constructed in the potential model shellfish species dwarf surf clam Mulinia lateralis using the ef1α gene promoter sequence, and the ptdTomato-ef1α2524 plasmid with high expression activity was screened through in vitro experiments. Subsequently, electroporation transfection was utilized to deliver the reporter plasmid into eggs of the dwarf surf clam. Electroporation parameters including voltage, pulse duration, pulse number, and plasmid mass concentration were optimized, and embryo-larval survival rates and transfection efficient were evaluated. It was found that the optimal electroporation were observed under conditions of seawater electroporation buffer, plasmid concentration 60 μg/mL, voltage 150 V, pulse duration 1 ms, and single pulse application. Under these conditions, there was transfection efficiency of (33.66±2.71)%. The results successfully realized efficient vector delivery into eggs of dwarf surf clam by electroporation, and established a shellfish-adapted electroporation technology platform. These findings provide robust technical support for molecular mechanism exploration, gene function studies, and breeding technology development in shellfish and other aquatic species, significantly promoting the advancement of gene editing technologies in mollusks.
[1] ROSENBERG G. A new critical estimate of named species-level diversity of the recent Mollusca[J]. American Malacological Bulletin,2014,32(2):308. [2] 张红会,朱爱意,王雪辉,等.珠海外伶仃海洋牧场生态系统服务价值评估研究[J].中国水运,2023,23(22):51-54. [3] KIM T K, EBERWINE J H. Mammalian cell transfection:the present and the future[J]. Analytical and Bioanalytical Chemistry,2010,397(8):3173-3178. [4] WANG S,ZHANG J B, JIAO W Q, et al. Scallop genome provides insights into evolution of bilaterian karyotype and development[J]. Nature Ecology & Evolution,2017,1:120. [5] WAGNER A, VORAUER-UHL K. Liposome technology for industrial purposes[J]. Journal of Drug Delivery,2011,2011(1):591325. [6] GRAESSMANN M, GRAESSMANN A. Microinjection of tissue culture cells[J]. Methods in Enzymology,1983,101:482-492. [7] KUMAR P, NAGARAJAN A, UCHIL P D. Electroporation[J]. Cold Spring Harbor Protocols,2019,2019(7):519-525. [8] SHENG Y L, MANCINO V, BIRREN B. Transformation of Escherichia coli with large DNA molecules by electroporation[J]. Nucleic Acids Research,1995,23(11):1990-1996. [9] GANDHI S, HAEUSSLER M, RAZY-KRAJKA F, et al. Evaluation and rational design of guide RNAs for efficient CRISPR/Cas9-mediated mutagenesis in Ciona[J]. Developmental Biology,2017,425(1):8-20. [10] SUN P S, VENZON N C, CALDERON F R O, et al. Evaluation of methods for DNA delivery into shrimp zygotes of Penaeus(Litopenaeus) vannamei[J]. Aquaculture,2005,243(1/2/3/4):19-26. [11] PRESTON N P, BAULE V J, LEOPOLD R, et al. Delivery of DNA to early embryos of the kuruma prawn, Penaeus japonicus[J]. Aquaculture,2000,181(3/4):225-234. [12] 于红,刘欣,李琪.基因编辑技术在贝类中的应用进展与展望[J].水产学报,2022,46(4):636-643. [13] 喻达辉,贾晓平,陈素文,等.合浦珠母贝卵子电击脉冲参数的初步研究[J].海洋科学,2005,29(8):15-19. [14] CHEN J, WU C L, ZHANG B L, et al. PiggyBac transposon-mediated transgenesis in the Pacific oyster (Crassostrea gigas) - first time in mollusks[J]. Frontiers in Physiology,2018,9:811. [15] CHAN J L, ZHANG W, XU Y, et al. Electroporation-based CRISPR/Cas9 mosaic mutagenesis of β-tubulin in the cultured oyster[J]. Frontiers in Marine Science,2022,9:912409. [16] YOON J, GU W B, KONUMA M, et al. Gene delivery available in molluscan cells by strong promoter discovered from bivalve-infectious virus[J]. Proceedings of the National Academy of Sciences of the United States of America,2022,119(45):e2209910119. [17] WANG Y H, ZHU X M, LIAN S S, et al. Functional characterization of Cfap206 for bivalve ciliogenesis by RNAi and CRISPR/Cas9 technologies[J]. Frontiers in Marine Science,2022,9:864037. [18] LIN J C, VAN EENENNAAM A L. Electroporation-mediated genome editing of livestock zygotes[J]. Frontiers in Genetics,2021,12:648482. [19] ESCOFFRE J M, PORTET T, FAVARD C, et al. Electromediated formation of DNA complexes with cell membranes and its consequences for gene delivery[J]. Biochimica et Biophysica Acta (BBA) - Biomembranes,2011,1808(6):1538-1543. [20] HASHIMOTO M, TAKEMOTO T. Electroporation enables the efficient mRNA delivery into the mouse zygotes and facilitates CRISPR/Cas9-based genome editing[J]. Scientific Reports,2015,5:11315. [21] 秦贞奎,杨祖晶,王浩,等.一种侏儒蛤的引种繁育方法:CN114027232A[P].2022-02-11. [22] YUE C Y, YU H, LI H J, et al. The endogenous EF-1α promoter is highly active in driving gene overexpression in developing embryos of the Pacific oyster Crassostrea gigas[J]. Aquaculture,2020,522:735134. [23] YU Y Z, LI Q, YU H, et al. Comparative analysis of promoter activity in Crassostrea gigas embryos:implications for bivalve gene editing[J]. Marine Biotechnology,2024,27(1):20. [24] GOWRISHANKAR T R, ESSER A T, VASILKOSKI Z, et al. Microdosimetry for conventional and supra-electroporation in cells with organelles[J]. Biochemical and Biophysical Research Communications,2006,341(4):1266-1276. [25] SAULIS G, SAULÉ R. Size of the pores created by an electric pulse:microsecond vs millisecond pulses[J]. Biochimica et Biophysica Acta (BBA) - Biomembranes,2012,1818(12):3032-3039. [26] JIN K D, ZHANG B L, JIN Q Q, et al. CRISPR/Cas9 system-mediated gene editing in the Fujian oysters (Crassostrea angulate) by electroporation[J]. Frontiers in Marine Science,2021,8:763470. [27] VIERRA D A, IRVINE S Q. Optimized conditions for transgenesis of the ascidian Ciona using square wave electroporation[J]. Development Genes and Evolution,2012,222(1):55-61. [28] PAKHOMOVA O N, GREGORY B W, KHOROKHORINA V A, et al. Electroporation-induced electrosensitization[J]. PLoS One,2011,6(2):e17100. [29] GOLZIO M, ROLS M P, TEISSIÉ J. In vitro and in vivo electric field-mediated permeabilization, gene transfer, and expression[J]. Methods,2004,33(2):126-135. [30] MEHIER-HUMBERT S, GUY R H. Physical methods for gene transfer:improving the kinetics of gene delivery into cells[J]. Advanced Drug Delivery Reviews,2005,57(5):733-753. [31] 赵日,蒙露,冯建琼,等.质粒电穿孔Jurkat细胞递送CRISPR/Cas9系统的优化[J].中国细胞生物学学报,2019,41(4):702-709. [32] LAMICHHANE T N, RAIKER R S, JAY S M. Exogenous DNA loading into extracellular vesicles via electroporation is size-dependent and enables limited gene delivery[J]. Molecular Pharmaceutics,2015,12(10):3650-3657. [33] XIE T D, SUN L, TSONG T Y. Study of mechanisms of electric field-induced DNA transfection. I. DNA entry by surface binding and diffusion through membrane pores[J]. Biophysical Journal,1990,58(1):13-19. [34] ZELLER R W. Electroporation in ascidians:history, theory and protocols[M]//SASAKURA Y. Transgenic Ascidians. Singapore:Springer Singapore,2018:37-48.
2025, Vol. 44 
