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Lentiviral vector transduction of spermatozoa as a tool for the study of early development.

Chandrashekran A, Isa I, Dudhia J, Thrasher AJ, Dibb N, Casimir C, Readhead C, Winston R - FEBS Open Bio (2014)

Bottom Line: Both can be genetically modified and used independently for the generation of transgenic animals or gene transfer/therapy of inherited disorders.Here we show that mature spermatozoa can be directly transduced with various pseudotyped lentiviral vectors and used in in vitro fertilisation studies.When these transduced sperm were used in in vitro fertilisation studies, GFP expression was observed in arising blastocysts.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery and Cancer, Division of Cancer, Imperial College London, Hammersmith Campus, Institute of Reproductive and Developmental Biology (IRDB), Du Cane Road, London W12 0NN, UK.

ABSTRACT
Spermatozoa and lentiviruses are two of nature's most efficient gene delivery vehicles. Both can be genetically modified and used independently for the generation of transgenic animals or gene transfer/therapy of inherited disorders. Here we show that mature spermatozoa can be directly transduced with various pseudotyped lentiviral vectors and used in in vitro fertilisation studies. Lentiviral vectors encoding Green Fluorescent Protein (GFP) were shown to be efficiently processed and expressed in sperm. When these transduced sperm were used in in vitro fertilisation studies, GFP expression was observed in arising blastocysts. This simple technique of directly transducing spermatozoa has potential to be a powerful tool for the study of early and pre-implantation development and could be used as a technique in transgenic development and vertical viral transmission studies.

No MeSH data available.


Related in: MedlinePlus

Lentiviral transduction efficiencies in mature spermatozoa. Porcine spermatozoa were cytospun onto microscope slides and stained with anti c-kit-FITC (Santa Cruz) conjugated antibody. (A) Spermatozoa expressing c-kit on the acrosome region. (B) Enhanced transduction of efficiency of spermatozoa was observed when pSCF was used in conjunction with VSV-g envelope compared to VSV-g alone, as analysed by flow cytometry. (C) Comparison of UCOE and PGK promoters on transduction efficiencies and expression levels in sperm at an MOI of 10.
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f0015: Lentiviral transduction efficiencies in mature spermatozoa. Porcine spermatozoa were cytospun onto microscope slides and stained with anti c-kit-FITC (Santa Cruz) conjugated antibody. (A) Spermatozoa expressing c-kit on the acrosome region. (B) Enhanced transduction of efficiency of spermatozoa was observed when pSCF was used in conjunction with VSV-g envelope compared to VSV-g alone, as analysed by flow cytometry. (C) Comparison of UCOE and PGK promoters on transduction efficiencies and expression levels in sperm at an MOI of 10.

Mentions: Spermatozoa have been reported to express c-kit [23], the receptor for stem cell factor (SCF) ligand. We confirmed this finding by immunostaining porcine spermatozoa and demonstrating distinct c-kit expression on the acrosome (Fig. 3A). Consequently, VSV-g-pseudotyped lentivirus particles produced in packaging cells expressing porcine membrane-bound SCF (pSCF-VSV-g) showed enhanced levels (about 3.5-fold) of transduction on porcine spermatozoa compared to lentivirus pseudotyped with VSV-g alone (Fig. 3B). Similar results were also obtained on transduction of murine spermatozoa using lentiviruses displaying murine mbSCF [24].


Lentiviral vector transduction of spermatozoa as a tool for the study of early development.

Chandrashekran A, Isa I, Dudhia J, Thrasher AJ, Dibb N, Casimir C, Readhead C, Winston R - FEBS Open Bio (2014)

Lentiviral transduction efficiencies in mature spermatozoa. Porcine spermatozoa were cytospun onto microscope slides and stained with anti c-kit-FITC (Santa Cruz) conjugated antibody. (A) Spermatozoa expressing c-kit on the acrosome region. (B) Enhanced transduction of efficiency of spermatozoa was observed when pSCF was used in conjunction with VSV-g envelope compared to VSV-g alone, as analysed by flow cytometry. (C) Comparison of UCOE and PGK promoters on transduction efficiencies and expression levels in sperm at an MOI of 10.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4048842&req=5

f0015: Lentiviral transduction efficiencies in mature spermatozoa. Porcine spermatozoa were cytospun onto microscope slides and stained with anti c-kit-FITC (Santa Cruz) conjugated antibody. (A) Spermatozoa expressing c-kit on the acrosome region. (B) Enhanced transduction of efficiency of spermatozoa was observed when pSCF was used in conjunction with VSV-g envelope compared to VSV-g alone, as analysed by flow cytometry. (C) Comparison of UCOE and PGK promoters on transduction efficiencies and expression levels in sperm at an MOI of 10.
Mentions: Spermatozoa have been reported to express c-kit [23], the receptor for stem cell factor (SCF) ligand. We confirmed this finding by immunostaining porcine spermatozoa and demonstrating distinct c-kit expression on the acrosome (Fig. 3A). Consequently, VSV-g-pseudotyped lentivirus particles produced in packaging cells expressing porcine membrane-bound SCF (pSCF-VSV-g) showed enhanced levels (about 3.5-fold) of transduction on porcine spermatozoa compared to lentivirus pseudotyped with VSV-g alone (Fig. 3B). Similar results were also obtained on transduction of murine spermatozoa using lentiviruses displaying murine mbSCF [24].

Bottom Line: Both can be genetically modified and used independently for the generation of transgenic animals or gene transfer/therapy of inherited disorders.Here we show that mature spermatozoa can be directly transduced with various pseudotyped lentiviral vectors and used in in vitro fertilisation studies.When these transduced sperm were used in in vitro fertilisation studies, GFP expression was observed in arising blastocysts.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery and Cancer, Division of Cancer, Imperial College London, Hammersmith Campus, Institute of Reproductive and Developmental Biology (IRDB), Du Cane Road, London W12 0NN, UK.

ABSTRACT
Spermatozoa and lentiviruses are two of nature's most efficient gene delivery vehicles. Both can be genetically modified and used independently for the generation of transgenic animals or gene transfer/therapy of inherited disorders. Here we show that mature spermatozoa can be directly transduced with various pseudotyped lentiviral vectors and used in in vitro fertilisation studies. Lentiviral vectors encoding Green Fluorescent Protein (GFP) were shown to be efficiently processed and expressed in sperm. When these transduced sperm were used in in vitro fertilisation studies, GFP expression was observed in arising blastocysts. This simple technique of directly transducing spermatozoa has potential to be a powerful tool for the study of early and pre-implantation development and could be used as a technique in transgenic development and vertical viral transmission studies.

No MeSH data available.


Related in: MedlinePlus