Limits...
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

Transgenic mouse blastocysts obtained following lentiviral transduction of mature spermatozoa. (A) Murine spermatozoa were incubated for 3 h with mSCF-VSV-g-pseudotyped lentiviral vector and subjected to confocal imaging. Middle panel: with transmitted light and bottom panel, without transmitted light showing intense GFP expression, particularly in the mid-piece and in the head. The top panel of this image was sperm not expressing GFP (untransduced), which was an image from a different field of view from the same chamber imaged using identical settings. GFP expression could only be observed using the 40× magnification. (B) Blastocysts obtained from mock transduced spermatozoa with transmitted light. (C) As in B without the transmitted light. (D) Fluorescent blastocysts obtained from lentivirally transduced spermatozoa, with transmitted light. (E) As in D without the transmitted light. All images were obtained using identical settings for fluorescence detection, in controls (mock) and transduced sperm/blastocysts.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4048842&req=5

f0030: Transgenic mouse blastocysts obtained following lentiviral transduction of mature spermatozoa. (A) Murine spermatozoa were incubated for 3 h with mSCF-VSV-g-pseudotyped lentiviral vector and subjected to confocal imaging. Middle panel: with transmitted light and bottom panel, without transmitted light showing intense GFP expression, particularly in the mid-piece and in the head. The top panel of this image was sperm not expressing GFP (untransduced), which was an image from a different field of view from the same chamber imaged using identical settings. GFP expression could only be observed using the 40× magnification. (B) Blastocysts obtained from mock transduced spermatozoa with transmitted light. (C) As in B without the transmitted light. (D) Fluorescent blastocysts obtained from lentivirally transduced spermatozoa, with transmitted light. (E) As in D without the transmitted light. All images were obtained using identical settings for fluorescence detection, in controls (mock) and transduced sperm/blastocysts.

Mentions: To further determine whether lentivirally transduced spermatozoa could be used for gene transfer to developing embryos; IVF experiments were carried out in mice. “Swim up” populations of mouse spermatozoa from the epididymis and vas deferens were obtained from 8–12 week old male mice and incubated at 32 °C with mSCF-VSV-g pseudotyped lentiviral vectors for 3 h prior to sperm imaging or IVF. Following lentiviral transduction, fluorescent mouse spermatozoa could also be visualised by confocal imaging (Fig. 6A, bottom two panels). Using standard IVF protocols, transduced sperm gave rise to transgenic blastocysts as shown in Fig. 6D and E. This figure is from the 3rd experiment carried out.


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)

Transgenic mouse blastocysts obtained following lentiviral transduction of mature spermatozoa. (A) Murine spermatozoa were incubated for 3 h with mSCF-VSV-g-pseudotyped lentiviral vector and subjected to confocal imaging. Middle panel: with transmitted light and bottom panel, without transmitted light showing intense GFP expression, particularly in the mid-piece and in the head. The top panel of this image was sperm not expressing GFP (untransduced), which was an image from a different field of view from the same chamber imaged using identical settings. GFP expression could only be observed using the 40× magnification. (B) Blastocysts obtained from mock transduced spermatozoa with transmitted light. (C) As in B without the transmitted light. (D) Fluorescent blastocysts obtained from lentivirally transduced spermatozoa, with transmitted light. (E) As in D without the transmitted light. All images were obtained using identical settings for fluorescence detection, in controls (mock) and transduced sperm/blastocysts.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

f0030: Transgenic mouse blastocysts obtained following lentiviral transduction of mature spermatozoa. (A) Murine spermatozoa were incubated for 3 h with mSCF-VSV-g-pseudotyped lentiviral vector and subjected to confocal imaging. Middle panel: with transmitted light and bottom panel, without transmitted light showing intense GFP expression, particularly in the mid-piece and in the head. The top panel of this image was sperm not expressing GFP (untransduced), which was an image from a different field of view from the same chamber imaged using identical settings. GFP expression could only be observed using the 40× magnification. (B) Blastocysts obtained from mock transduced spermatozoa with transmitted light. (C) As in B without the transmitted light. (D) Fluorescent blastocysts obtained from lentivirally transduced spermatozoa, with transmitted light. (E) As in D without the transmitted light. All images were obtained using identical settings for fluorescence detection, in controls (mock) and transduced sperm/blastocysts.
Mentions: To further determine whether lentivirally transduced spermatozoa could be used for gene transfer to developing embryos; IVF experiments were carried out in mice. “Swim up” populations of mouse spermatozoa from the epididymis and vas deferens were obtained from 8–12 week old male mice and incubated at 32 °C with mSCF-VSV-g pseudotyped lentiviral vectors for 3 h prior to sperm imaging or IVF. Following lentiviral transduction, fluorescent mouse spermatozoa could also be visualised by confocal imaging (Fig. 6A, bottom two panels). Using standard IVF protocols, transduced sperm gave rise to transgenic blastocysts as shown in Fig. 6D and E. This figure is from the 3rd experiment carried out.

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