Dimethyl sulphoxide and electrolyte-free medium improve exogenous DNA uptake in mouse sperm and subsequently gene expression in the embryo

Kurd, Soleiman; Hosseini, Sara; Fathi, Fardin; Jajarmi, Vahid; Salehi, Mohammad

doi:10.1017/s0967199418000436

Cited by 4 publications

(5 citation statements)

References 25 publications

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“…These findings are in agreement with Kurd et al 's study, which hypothesized that cold shock causes the joining of environmental ions to the plasma membrane and causes changes in the membrane. They were able to reduce the detrimental effects of cold shock and DMSO on sperm functional parameters through incubation of mouse spermatozoa in an electrolyte-free medium (0.33 M glucose and 4 mg/ml BSA), resulting in improved viability, progressive motility, and an increase in the production rate of transgenic blastocysts obtained from the DMSO-SMGT method [52]. It is well documented that cold shock leads to irreversible suppression of motility and metabolic activities, disruption of the plasma and acrosome membranes, and increased permeability of spermatozoa of different species.…”

Section: Treatmentsmentioning

confidence: 99%

Methyl β-Cyclodextrin-sperm-mediated gene editing (MBCD-SMGE): a simple and efficient method for targeted mutant mouse production

Moradbeigi,

Hosseini,

Salehi

et al. 2024

Biol Proced Online

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Background Generating targeted mutant mice is a crucial technology in biomedical research. This study focuses on optimizing the CRISPR/Cas9 system uptake into sperm cells using the methyl β-cyclodextrin-sperm-mediated gene transfer (MBCD-SMGT) technique to generate targeted mutant blastocysts and mice efficiently. Additionally, the present study elucidates the roles of cholesterol and reactive oxygen species (ROS) in the exogenous DNA uptake by sperm. Results In this study, B6D2F1 mouse sperm were incubated in the c-TYH medium with different concentrations of MBCD (0, 0.75, 1, and 2 mM) in the presence of 20 ng/µl pCAG-eCas9-GFP-U6-gRNA (pgRNA-Cas9) for 30 min. Functional parameters, extracellular ROS, and the copy numbers of internalized plasmid per sperm cell were evaluated. Subsequently, in vitro fertilization (IVF) was performed and fertilization rate, early embryonic development, and transfection rate were assessed. Finally, our study investigated the potential of the MBCD-SMGT technique in combination with the CRISPR-Cas9 system, referred to as MBCD-SMGE (MBCD-sperm-mediated gene editing), for generating targeted mutant blastocysts and mice. Results indicated that cholesterol removal from the sperm membrane using MBCD resulted in a premature acrosomal reaction, an increase in extracellular ROS levels, and a dose-dependent influence on the copy numbers of the internalized plasmids per sperm cell. Moreover, the MBCD-SMGT technique led to a larger population of transfected motile sperm and a higher production rate of GFP-positive blastocysts. Additionally, the current study validated the targeted indel in blastocyst and mouse derived from MBCD-SMGE technique. Conclusion Overall, this study highlights the significant potential of the MBCD-SMGE technique for generating targeted mutant mice. It holds enormous promise for modeling human diseases and improving desirable traits in animals. Graphical Abstract

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Section: Treatmentsmentioning

confidence: 99%

Methyl β-Cyclodextrin-sperm-mediated gene editing (MBCD-SMGE): a simple and efficient method for targeted mutant mouse production

Moradbeigi,

Hosseini,

Salehi

et al. 2024

Biol Proced Online

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“…In SMGT method, a study by Kurd et al. (2018) showed that gene transfer using DMSO resulted in 62% of blastocysts having the transgene. In boars, 61.9% of sperms calls showed GFP positive expression (García‐Vázquez et al., 2011).…”

Section: Resultsmentioning

confidence: 99%

“…DMSO is known to have the ability to make temporary pores in the cell membrane, which can play a role in the uptake of the foreign DNA into target cells (Gironi et al, 2020;Kuznetsov et al, 2000). Several studies have reported the use of DMSO either in combination with other transfection methods or alone for purposes of entering DNA into sperm cells with the best cellular uptake (Collares et al, 2011;Kurd et al, 2018;Kuznetsov et al, 2000;Shen et al, 2006). Amaral et al (2011) used plasmid DNA (pEGFP-N1) mixed with DMSO, DMA, or liposome (Lipofectin) for testis gene transfer.…”

Section: Dmsomentioning

confidence: 99%

“…Hassanane et al (2017), used DMSO to transfer the construct (pEGFP-N1) onto the sperm of buffalo (SMGT), and the expression of GFP was observed in 74.2% of Blastocysts (Hassanane et al, 2017). In SMGT method, a study by Kurd et al (2018) showed that gene transfer using DMSO resulted in 62% of blastocysts having the transgene. In boars, 61.9% of sperms calls showed GFP positive expression (García-Vázquez et al, 2011).…”

Section: Dmsomentioning

confidence: 99%

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Comparison of two methods of sperm‐ and testis‐mediated gene transfer in production of transgenic animals: A systematic review

Dehghan,

Darya,

Mehdinejadiani

et al. 2024

Animal Genetics

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Transgenic (Tg) animal technology is one of the growing areas in biology. Various Tg technologies, each with its own advantages and disadvantages, are available for generating Tg animals. These include zygote microinjection, electroporation, viral infection, embryonic stem cell or spermatogonial stem cell‐mediated production of Tg animals, sperm‐mediated gene transfer (SMGT), and testis‐mediated gene transfer (TMGT). However, there are currently no comprehensive studies comparing SMGT and TMGT methods, selecting appropriate gene delivery carriers (such as nanoparticles and liposomes), and determining the optimal route for gene delivery (SMGT and TMGT) for producing Tg animal. Here we aim to provide a comprehensive assessment comparing SMGT and TMGT methods, and to introduce the best carriers and gene transfer methods to sperm and testis to generate Tg animals in different species. From 2010 to 2022, 47 studies on SMGT and 25 studies on TMGT have been conducted. Mice and rats were the most commonly used species in SMGT and TMGT. Regarding the SMGT approach, nanoparticles, streptolysin‐O, and virus packaging were found to be the best gene transfer methods for generating Tg mice. In the TMGT method, the best gene transfer methods for generating Tg mice and rats were virus packaging, dimethyl sulfoxide, electroporation, and liposome. Our study has shown that the efficiency of producing Tg animals varies depending on the species, gene carrier, and method of gene transfer.

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“…Subsequent studies incorporating DMSO demonstrated a 42% transfection rate in embryos, accompanied by an accelerated progression toward the blastocyst stage and the production of GFP-positive blastocysts 33 . The application of the SMGT method notably accelerated the progression to the blastocyst stage and resulted in an increased production of GFP-positive blastocysts 34 . There is limited competition in the development of materials for enhancing transfection e ciency.…”

Section: Discussionmentioning

confidence: 99%

Simulating the effects of H2O2 and melatonin treatments on the oxidation and reduction state of sperm on two outcomes: DNA uptake in sperm and apoptosis of embryonic cells

Saberi,

Jajarmi,

Hosseini

et al. 2023

Preprint

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Improving the transfer of foreign genes into recalcitrant cells, as in the production of transgenic animal models, is a challenge. Sperm-mediated gene transfer (SMGT) is a cost-effective and rapid method for exogenous DNA transferring into egg by sperm during fertilization. This method can be improved using chemical and natural additives. The objective of this study was to examine how sperm function, DNA uptake, embryonic development, and apoptosis rates are affected by different levels of ROS under varying oxidant and antioxidant conditions in SMGT method. Sperm isolated from mice with and without plasmid were treated with varying concentrations of H2O2 and melatonin. Under oxidation conditions, sperm performance was significantly reduced compared to the melatonin treatment. Embryos from the oxidation group with 100 µM H2O2 treatment showed higher uptake of exogenous DNA, and the proportion of GFP-positive blastocysts was significantly higher than those from higher concentrations. The data demonstrate that oxidative conditions in sperm are associated with increased absorption of exogenous DNA, potentially due to an increase in the mitochondria ability resulting from reactive oxygen species increase and sperm membrane instability. The importance of antioxidants lies in their ability to safeguard DNA against oxidative stress, enhance absorption efficacy, and bolster the stability of the sperm membrane. The study showed that antioxidants reduced the risk of negative effects on sperm quality and function.

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Dimethyl sulphoxide and electrolyte-free medium improve exogenous DNA uptake in mouse sperm and subsequently gene expression in the embryo

Cited by 4 publications

References 25 publications

Methyl β-Cyclodextrin-sperm-mediated gene editing (MBCD-SMGE): a simple and efficient method for targeted mutant mouse production

Methyl β-Cyclodextrin-sperm-mediated gene editing (MBCD-SMGE): a simple and efficient method for targeted mutant mouse production

Comparison of two methods of sperm‐ and testis‐mediated gene transfer in production of transgenic animals: A systematic review

Simulating the effects of H2O2 and melatonin treatments on the oxidation and reduction state of sperm on two outcomes: DNA uptake in sperm and apoptosis of embryonic cells

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