Genomics and Applied Biology 2024, Vol.15, No.2, 89-98 http://bioscipublisher.com/index.php/gab 90 2 Background on Shrimp Genetics 2.1 Genetic structure and diversity in shrimp Shrimp species exhibit significant genetic diversity, which is crucial for their adaptability and survival in various environments. For instance, the Pacific white shrimp (Litopenaeus vannamei) has been extensively studied for its genetic diversity using techniques such as microsatellite markers. These markers have revealed high levels of heterozygosity and numerous alleles specific to different populations, indicating a rich genetic variation within the species (Wolfus et al., 1997). Additionally, the development of high-throughput SNP genotyping methods has further enhanced our understanding of genetic relationships and diversity in shrimp populations, facilitating more efficient breeding programs (Yu et al., 2020). 2.2 Challenges in shrimp genetic manipulation Despite the advances in genetic studies, manipulating shrimp genetics presents several challenges. One major issue is the efficient delivery and stable integration of foreign genes into shrimp genomes. Traditional methods like microinjection and electroporation have shown limited success, with low hatching rates and gene expression levels (Sun et al., 2005). Moreover, the presence of mitochondrial pseudogenes, which can be mistaken for functional genes, complicates genetic analyses and manipulations (Williams and Knowlton, 2001). The development of more effective gene transfer techniques, such as the use of nuclear localization signals (NLS) to enhance nuclear import of vector DNA, has shown promise but still requires further optimization (Arenal et al., 2004). 2.3 Historical overview of gene transfer in shrimp The history of gene transfer in shrimp has seen gradual improvements in techniques and outcomes. Early methods focused on microinjection and electroporation, which had limited success due to low efficiency and high mortality rates. The introduction of transfection reagents significantly improved gene transfer efficiency, with studies reporting up to 60% hatching rates and 72% gene transfer efficiency when using DNA/jetPEI complexes (Sun et al., 2005). More recently, the development of a VP28-pseudotyped baculovirus expression system has achieved near 100% infection efficiency in specific tissues, marking a significant advancement in the field (Wu et al., 2021). These historical developments highlight the ongoing efforts to refine gene transfer techniques to enhance their applicability and success in shrimp genetic manipulation. 3 Gene Transfer Techniques in Shrimp 3.1 Overview of common gene transfer methods Gene transfer techniques are essential tools in shrimp genetic research, enabling the study and manipulation of genes to understand their functions and improve shrimp breeding programs. The most common methods include microinjection, electroporation, lipofection, and viral vectors. 3.1.1 Microinjection Microinjection is a direct and reliable method for gene transfer, widely used in various aquatic species. This technique involves injecting nucleic acids, gene constructs, or other substances directly into shrimp embryos or cells using fine needles. It has been extensively used in fish and other aquatic organisms due to its precision and effectiveness (Abdelrahman et al., 2021; Lane et al., 2021; Gultom, 2023). Despite requiring sophisticated equipment and skilled personnel, microinjection remains a preferred method due to its high success rate and the ability to deliver precise amounts of genetic material (Takahashi et al., 2015; Gu et al., 2018). 3.1.2 Electroporation Electroporation involves applying an electrical field to cells to increase the permeability of the cell membrane, allowing nucleic acids to enter the cells. This method is less invasive than microinjection and can be used to transfect a large number of cells simultaneously. It has been successfully applied in various species, including mice, where it has shown effectiveness in delivering CRISPR/Cas9 components (Takahashi et al., 2015). Electroporation is advantageous for its simplicity and ability to handle large-scale gene transfer experiments.
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