Methods for Sperm-Mediated Gene Transfer

Lavitrano, Marialuisa; Giovannoni, Roberto; Cerrito, Maria Grazia

doi:10.1007/978-1-62703-038-0_44

Cited by 19 publications

(7 citation statements)

References 41 publications

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“…This vector could be used to produce human E5NT and ENTPD1-double transgenic pigs by a single round of Somatic Cell Nuclear Transfer (SCNT), [8] or by Sperm Mediated Gene Transfer (SMGT). [9] An increased production of adenosine could protect xenograft's endothelium against pro-inflammatory, pro-thrombotic, and immunological effects of ATP and ADP thanks to its anti-inflammatory, anti-thrombotic, and immunosuppressive properties. [4,5] This cell model, we established, could be useful for in vitro studies of these mechanisms.…”

Section: Resultsmentioning

confidence: 99%

Functional Analysis of Expression of Human Ecto-Nucleoside Triphosphate Diphosphohydrolase-1 and/or Ecto-5′-Nucleotidase in Pig Endothelial Cells

Giorgi

Pelikant‐Małecka

Sielicka

et al. 2014

Nucleosides, Nucleotides and Nucleic Acids

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Adenine nucleosides and nucleotides are important signaling molecules involved in control of key mechanisms of xenotransplant rejection. Extracellular pathway that converts ATP and ADP to AMP, and AMP to adenosine mainly mediated by ecto-nucleoside triphosphate diphosphohydrolase 1, (ENTPD1 or CD39) and ecto-5'-nucleotidase (E5NT or CD73) respectively, is considered as important target for xenograft protection. To clarify feasibility of combined expression of human ENTPD1 and E5NT and to study its functional effect we transfected pig endothelial cell line (PIEC) with both genes together. To do this we have produced a dicistronic construct bearing F2A sequence in frame between human E5NT and human ENTPD1 coding sequences. PIEC cells were mock-transfected as transfection control or transfected with plasmids encoding human ENTPD1 or human E5NT. PIEC cells were exposed to 50 μM ATP or 50 μM ADP or 50 μM AMP. Conversion of extracellular substrates into products (ATP/ADP/AMP/adenosine) was measured by HPLC in the media collected at specific time intervals. Following addition of AMP, production of adenosine in the medium of E5NT/ENTPD1- and E5NT- transfected cells increased to 14.2±1.1 and 24.5±3.4 μM respectively while it remained below 1 μM in controls and in ENTPD1-transfected cells. A marked increase of adenosine formation from ADP or ATP was observed only in E5NT/ENTPD1-transfected cells (11.7±0.1 and 5.7±2.2 μM respectively) but not in any other condition studied. This study indicates feasibility and functionality of combined expression of human E5NT and ENTPD1 in pig endothelial cells using F2A sequence bearing construct.

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Section: Resultsmentioning

confidence: 99%

Functional Analysis of Expression of Human Ecto-Nucleoside Triphosphate Diphosphohydrolase-1 and/or Ecto-5′-Nucleotidase in Pig Endothelial Cells

Giorgi

Pelikant‐Małecka

Sielicka

et al. 2014

Nucleosides, Nucleotides and Nucleic Acids

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“…Interestingly, in the control incubated with exogenous DNA, an increase in plasma membrane damage (~10%) was also evidenced with respect to the control without DNA incubation. This effect can be caused by the presence of exogenous DNA and manipulation of spermatozoa in the laboratory (Lavitrano et al ., 2013). In relation to the state of the acrosomal membrane, significant differences were observed between the different treatments and controls, in which treatments with LL and TX-100 showed a greater effect, with 100% of the spermatozoa showing positive reaction to the PNA/FITC probe, unlike the treatment with NaOH, which showed a lower proportion (87%).…”

Section: Discussionmentioning

confidence: 99%

“…Sperm-mediated gene transfer (SMGT) is a simple biotechnological tool that takes advantage of the ability of spermatozoa to bind, internalize, and transport exogenous molecules within the oocyte during fertilization, allowing the generation of embryos and/or transgenic offspring (Lavitrano et al ., 2013). However, the efficiency of this technique remains low mainly due to a low level of binding of exogenous DNA in the spermatozoa.…”

Section: Introductionmentioning

confidence: 99%

Improved exogenous DNA uptake in bovine spermatozoa and gene expression in embryos using membrane destabilizing agents in ICSI-SMGT

Sánchez-Villalba

Arias

Zambrano

et al. 2018

Zygote

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Sperm-mediated gene transfer (SMGT) is a simple, fast, and economical biotechnological tool for producing transgenic animals. However, transgene expression with this technique in bovine embryos is still inefficient due to low uptake and binding of exogenous DNA in spermatozoa. The present study evaluated the effects of sperm membrane destabilization on the binding capacity, location and quantity of bound exogenous DNA in cryopreserved bovine spermatozoa using Triton X-100 (TX-100), lysolecithin (LL) and sodium hydroxide (NaOH). Effects of these treatments were also evaluated by intracytoplasmic sperm injection (ICSI)-SMGT. Results showed that all treatments bound exogenous DNA to spermatozoa including the control. Spermatozoa treated with different membrane destabilizing agents bound the exogenous DNA throughout the head and tail of spermatozoa, compared with the control, in which binding occurred mainly in the post-acrosomal region and tail. The amount of exogenous DNA bound to spermatozoa was much higher for the different sperm treatments than the control (P < 0.05), most likely due to the damage induced by these treatments to the plasma and acrosomal membranes. Exogenous gene expression in embryos was also improved by these treatments. These results demonstrated that sperm membrane destabilization could be a novel strategy in bovine SMGT protocols for the generation of transgenic embryos by ICSI.

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“…In addition to lentiviral mediated gene transfer, sperm mediated gene transfer (SMGT) developed in the 1980s has been attempted to create transgenic NHPs (Chan et al 2000a;Chan et al 2000b;Chan et al 2000c). Instead of co-incubation of sperm with naked DNA, followed by artificial insemination in domestic species (Lavitrano et al 1989;Lavitrano et al 2013), NHP sperm were incubated with plasmid DNA, followed by intracytoplasmic sperm injection (ICSI) (Chan et al 2000a;Chan et al 2000c). Although this approach did not result in transgenic NHPs, a similar approach has been successfully used for creating transgenic mice (Perry et al 1999).…”

Section: Introductionmentioning

confidence: 99%

Progress and Prospects for Genetic Modification of Nonhuman Primate Models in Biomedical Research

Chan¹

2013

ILAR Journal

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The growing interest of modeling human diseases using genetically modified (transgenic) nonhuman primates (NHPs) is a direct result of NHPs (rhesus macaque, etc.) close relation to humans. NHPs share similar developmental paths with humans in their anatomy, physiology, genetics, and neural functions; and in their cognition, emotion, and social behavior. The NHP model within biomedical research has played an important role in the development of vaccines, assisted reproductive technologies, and new therapies for many diseases. Biomedical research has not been the primary role of NHPs. They have mainly been used for safety evaluation and pharmacokinetics studies, rather than determining therapeutic efficacy. The development of the first transgenic rhesus macaque (2001) revolutionized the role of NHP models in biomedicine. Development of the transgenic NHP model of Huntington's disease (2008), with distinctive clinical features, further suggested the uniqueness of the model system; and the potential role of the NHP model for human genetic disorders. Modeling human genetic diseases using NHPs will continue to thrive because of the latest advances in molecular, genetic, and embryo technologies. NHPs rising role in biomedical research, specifically pre-clinical studies, is foreseeable. The path toward the development of transgenic NHPs and the prospect of transgenic NHPs in their new role in future biomedicine needs to be reviewed. This article will focus on the advancement of transgenic NHPs in the past decade, including transgenic technologies and disease modeling. It will outline new technologies that may have significant impact in future NHP modeling and will conclude with a discussion of the future prospects of the transgenic NHP model.

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Methods for Sperm-Mediated Gene Transfer

Cited by 19 publications

References 41 publications

Functional Analysis of Expression of Human Ecto-Nucleoside Triphosphate Diphosphohydrolase-1 and/or Ecto-5′-Nucleotidase in Pig Endothelial Cells

Functional Analysis of Expression of Human Ecto-Nucleoside Triphosphate Diphosphohydrolase-1 and/or Ecto-5′-Nucleotidase in Pig Endothelial Cells

Improved exogenous DNA uptake in bovine spermatozoa and gene expression in embryos using membrane destabilizing agents in ICSI-SMGT

Progress and Prospects for Genetic Modification of Nonhuman Primate Models in Biomedical Research

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