Protamine-Derived Synthetic Peptide Enhances the Efficiency of Sperm-Mediated Gene Transfer Using Liposome-Peptide-DNA Complex.

Yonezawa, Tomohiro; Furuhata, Yasufumi; Hirabayashi, Keiji; Suzuki, Masatoshi; Yamanouchi, Keitaro; Nishihara, Masugi

doi:10.1262/jrd.48.281

Cited by 8 publications

(3 citation statements)

References 30 publications

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“…8 Similar results have also shown a lack of uptake of foreign DNA by rodent spermatozoa in the absence of lipid carriers. 14 The current study’s findings further confirm that liposomal carriers facilitate the movement of foreign genes across the cell membranes of host cells and incorporation into the host cell genome.…”

Section: Discussionsupporting

confidence: 75%

Comparative Efficiency for in vitro Transfection of Goat Undifferentiated Spermatogonia Using Lipofectamine Reagents and Electroporation

Nakami¹,

Nguhiu-Mwangi²,

Kipyegon³

et al. 2022

SCCAA

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Introduction Spermatogonial stem cells (SSC), also referred to as undifferentiated spermatogonia, are the germline stem cells responsible for continuous spermatogenesis throughout a male’s life. They are, therefore, an ideal target for gene editing. Previously, SSC from animal testis have been isolated and transplanted to homologous recipients resulting in the successful reestablishment of donor-derived spermatogenesis. Methods Enhanced green fluorescent protein (eGFP) gene transfection into goat SSC was evaluated using liposomal carriers and electroporation. The cells were isolated from the prepubertal Galla goats testis cultured in serum-free defined media and transfected with the eGFP gene. Green fluorescing of SSC colonies indicated transfection. Results The use of lipofectamine TM stem reagent and lipofectamine TM 2000 carriers resulted in more SSC colonies expressing the eGFP gene (25.25% and 22.25%, respectively). Electroporation resulted in 15% ± 0.54 eGFP expressing SSC colonies. Furthermore, cell viability was higher in lipofectamine transfection (55% ± 0.21) as compared to electroporation (38% ± 0.14). Conclusion These results indicated that lipofectamine was more effective in eGFP gene transfer into SSC. The successful transient transfection points to a possibility of transfecting transgenes into male germ cells in genetic engineering programs.

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Section: Discussionsupporting

confidence: 75%

Comparative Efficiency for in vitro Transfection of Goat Undifferentiated Spermatogonia Using Lipofectamine Reagents and Electroporation

Nakami¹,

Nguhiu-Mwangi²,

Kipyegon³

et al. 2022

SCCAA

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“…From this study, it was suggested that SMGT-AI can be successfully adapted for the generation of Tg livestock. Yonezawa et al [99] tested whether liposome-peptide (derived from human protamine)-DNA complex (LPD), a new reagent known to stabilize transfection in cultured cells, was useful to increase the efficiency of SMGT. They performed AI using rat epididymal sperm that had been incubated in a solution containing GFP expressing plasmid DNA and LPD complex.…”

Section: Historical Background Of Smgt-based Aimentioning

confidence: 99%

Possible Production of Genome-Edited Animals Using Gene-Engineered Sperm

Nakamura¹

2019

Gene Editing - Technologies and Applications

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CRISPR/Cas9 is widely used for genome editing in a variety of organisms, including mammals, fishes, and plants. In mammals, zygotes are considered an appropriate target for gene delivery of CRISPR/Cas9 components [Cas9 endonuclease and a single-guide (sgRNA)] via microinjection or in vitro electroporation. However, these approaches require ex vivo handling of zygotes, which is necessary for egg transfer to recipient females to allow the treated zygotes to develop fullterm. These procedures are often laborious, time-consuming, and use numerous mice. In our previous experiments, the plasmid DNA encapsulated by liposomal reagent introduced into the internal portion of a testis can be transferred to the mature sperm present in the epididymal ducts, and is finally transferred to oocytes via fertilization. Although it was not integrated into their genome, this approach would be useful for creating genome-edited animals, since CRISPR/Cas9 can be performed by transient interaction of Cas9 and sgRNA, whereby chromosomal integration of the CRISPR components is not a prerequisite. Here, we will review past achievements concerning in vivo transfection of immature/mature sperm and present experimental proposals for possible genome editing via gene-engineered sperm based on recent findings.

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“…This process, termed sperm-mediated gene transfer (SMGT), provides a simple and straightforward method to produce transgenic animals. However, the SMGT protocol has been extended to many animal species, including mice [3] , [4] , rats [5] , rabbits [6] , and pigs [7] , [8] . It has also been a controversial issue in the past two decades as several other groups reported failure in repeat the original gene transfer protocol [9] .…”

Section: Introductionmentioning

confidence: 99%

Production of Transgenic Pigs Mediated by Pseudotyped Lentivirus and Sperm

Zhang

Ding

et al. 2012

PLoS ONE

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Sperm-mediated gene transfer can be a very efficient method to produce transgenic pigs, however, the results from different laboratories had not been widely repeated. Genomic integration of transgene by injection of pseudotyped lentivirus to the perivitelline space has been proved to be a reliable route to generate transgenic animals. To test whether transgene in the lentivirus can be delivered by sperm, we studied incubation of pseudotyped lentiviruses and sperm before insemination. After incubation with pig spermatozoa, 62±3 lentiviral particles were detected per 100 sperm cells using quantitative real-time RT-PCR. The association of lentivirus with sperm was further confirmed by electron microscopy. The sperm incubated with lentiviral particles were artificially inseminated into pigs. Of the 59 piglets born from inseminated 5 sows, 6 piglets (10.17%) carried the transgene based on the PCR identification. Foreign gene and EGFP was successfully detected in ear tissue biopsies from two PCR-positive pigs, revealed via in situ hybridization and immunohistochemistry. Offspring of one PCR-positive boar with normal sows showed PCR-positive. Two PCR-positive founders and offsprings of PCR-positive boar were further identified by Southern-blot analysis, out of which the two founders and two offsprings were positive in Southern blotting, strongly indicating integration of foreign gene into genome. The results indicate that incubation of sperm with pseudotyped lentiviruses can incorporated with sperm-mediated gene transfer to produce transgenic pigs with improved efficiency.

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Protamine-Derived Synthetic Peptide Enhances the Efficiency of Sperm-Mediated Gene Transfer Using Liposome-Peptide-DNA Complex.

Cited by 8 publications

References 30 publications

Comparative Efficiency for in vitro Transfection of Goat Undifferentiated Spermatogonia Using Lipofectamine Reagents and Electroporation

Comparative Efficiency for in vitro Transfection of Goat Undifferentiated Spermatogonia Using Lipofectamine Reagents and Electroporation

Possible Production of Genome-Edited Animals Using Gene-Engineered Sperm

Production of Transgenic Pigs Mediated by Pseudotyped Lentivirus and Sperm

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