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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

Validation of GFP expression in spermatozoa. (A)Porcine spermatozoa were transfected with si RNA to GFP or scrambled/control siRNA (10 nM) using Escort V (Sigma) in triplicate. Two hours later, VSV-g-pSCF pseudotyped lentiviral vectors were added to the siRNA transfected cells and incubation carried out for a further 24 h. GFP expression was then compared by flow cytometery. (B) Porcine spermatozoa were initially incubated with 1 μM AZT in triplicate. Again 2 h later, VSV-g-pSCF pseudotyped lentiviral vectors were added to the sperm culture and GFP expression determined by flow cytomtery 24 h later. (C) Porcine spermatozoa were incubated with a VSV-g-pSCF pseudotyped lentiviral vector at two different temperatures. GFP expression was again determined 24 h later by flow cytometry.
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f0020: Validation of GFP expression in spermatozoa. (A)Porcine spermatozoa were transfected with si RNA to GFP or scrambled/control siRNA (10 nM) using Escort V (Sigma) in triplicate. Two hours later, VSV-g-pSCF pseudotyped lentiviral vectors were added to the siRNA transfected cells and incubation carried out for a further 24 h. GFP expression was then compared by flow cytometery. (B) Porcine spermatozoa were initially incubated with 1 μM AZT in triplicate. Again 2 h later, VSV-g-pSCF pseudotyped lentiviral vectors were added to the sperm culture and GFP expression determined by flow cytomtery 24 h later. (C) Porcine spermatozoa were incubated with a VSV-g-pSCF pseudotyped lentiviral vector at two different temperatures. GFP expression was again determined 24 h later by flow cytometry.

Mentions: To validate that the fluorescence observed in porcine spermatozoa was due to lentiviral encoded, de novo GFP synthesis, we performed experiments to block the expression of GFP, either with the HIV reverse transcriptase inhibitor, azidodeoxy thimidine (AZT) (Fig. 4B), or by transfection (Escort V, Sigma) with silencing RNA to GFP (siRNA-GFP) (Ambion) (Fig. 4A). When sperm were incubated with 1 μM AZT or 10 nM siRNA-GFP 2 h prior to the addition of the lentiviral vector, GFP expression, as determined by flow cytometry, was inhibited by about 50% compared to untreated or scrambled siRNA controls (Fig. 4A and B). Furthermore, when porcine spermatozoa were incubated with lentiviral vectors at 18 °C for 24 h, GFP expression was reduced by at least 3-fold compared to incubations carried out at 32 °C (Fig. 4C), demonstrating that the GFP expression in mature spermatozoa was dependent on lentiviral transduction.


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)

Validation of GFP expression in spermatozoa. (A)Porcine spermatozoa were transfected with si RNA to GFP or scrambled/control siRNA (10 nM) using Escort V (Sigma) in triplicate. Two hours later, VSV-g-pSCF pseudotyped lentiviral vectors were added to the siRNA transfected cells and incubation carried out for a further 24 h. GFP expression was then compared by flow cytometery. (B) Porcine spermatozoa were initially incubated with 1 μM AZT in triplicate. Again 2 h later, VSV-g-pSCF pseudotyped lentiviral vectors were added to the sperm culture and GFP expression determined by flow cytomtery 24 h later. (C) Porcine spermatozoa were incubated with a VSV-g-pSCF pseudotyped lentiviral vector at two different temperatures. GFP expression was again determined 24 h later by flow cytometry.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4048842&req=5

f0020: Validation of GFP expression in spermatozoa. (A)Porcine spermatozoa were transfected with si RNA to GFP or scrambled/control siRNA (10 nM) using Escort V (Sigma) in triplicate. Two hours later, VSV-g-pSCF pseudotyped lentiviral vectors were added to the siRNA transfected cells and incubation carried out for a further 24 h. GFP expression was then compared by flow cytometery. (B) Porcine spermatozoa were initially incubated with 1 μM AZT in triplicate. Again 2 h later, VSV-g-pSCF pseudotyped lentiviral vectors were added to the sperm culture and GFP expression determined by flow cytomtery 24 h later. (C) Porcine spermatozoa were incubated with a VSV-g-pSCF pseudotyped lentiviral vector at two different temperatures. GFP expression was again determined 24 h later by flow cytometry.
Mentions: To validate that the fluorescence observed in porcine spermatozoa was due to lentiviral encoded, de novo GFP synthesis, we performed experiments to block the expression of GFP, either with the HIV reverse transcriptase inhibitor, azidodeoxy thimidine (AZT) (Fig. 4B), or by transfection (Escort V, Sigma) with silencing RNA to GFP (siRNA-GFP) (Ambion) (Fig. 4A). When sperm were incubated with 1 μM AZT or 10 nM siRNA-GFP 2 h prior to the addition of the lentiviral vector, GFP expression, as determined by flow cytometry, was inhibited by about 50% compared to untreated or scrambled siRNA controls (Fig. 4A and B). Furthermore, when porcine spermatozoa were incubated with lentiviral vectors at 18 °C for 24 h, GFP expression was reduced by at least 3-fold compared to incubations carried out at 32 °C (Fig. 4C), demonstrating that the GFP expression in mature spermatozoa was dependent on lentiviral transduction.

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