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

Schematic map of the lentiviral vectors utilised in this study. (A) This vector is self-inactivating in transduced cells due to deletions of the enhancer region in the 3′ LTR. The reporter gene, GFP is driven by a housekeeping promoter PGK (top panel) and UCOE (bottom panel). (B) Flow cytometry analysis of porcine transduced spermatozoa. Porcine spermatozoa were incubated with a pSCF-VSV-g pseudotyped lentiviral vectors (Pgk promoter) and 7-AAD staining to exclude dead cells. (Top left panel) Mock/untransduced sperm light scatter analysis. (Top right panel) 7-AAD staining for exclusion of dead cells. (Bottom left panel) Untransduced (mock) live spermatozoa with no GFP expression. (Bottom right panel) Live and transduced spermatozoa showing 38% of spermatozoa expressing GFP.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

f0010: Schematic map of the lentiviral vectors utilised in this study. (A) This vector is self-inactivating in transduced cells due to deletions of the enhancer region in the 3′ LTR. The reporter gene, GFP is driven by a housekeeping promoter PGK (top panel) and UCOE (bottom panel). (B) Flow cytometry analysis of porcine transduced spermatozoa. Porcine spermatozoa were incubated with a pSCF-VSV-g pseudotyped lentiviral vectors (Pgk promoter) and 7-AAD staining to exclude dead cells. (Top left panel) Mock/untransduced sperm light scatter analysis. (Top right panel) 7-AAD staining for exclusion of dead cells. (Bottom left panel) Untransduced (mock) live spermatozoa with no GFP expression. (Bottom right panel) Live and transduced spermatozoa showing 38% of spermatozoa expressing GFP.

Mentions: Furthermore, when the VSV-g pseudotyped lentiviral vector encoding Histone 2B-fused Green Fluorescent Protein (H2B-GFP) (Fig. 2A top panel) was used to transduce 293T cells and porcine spermatozoa, newly synthesised GFP could be immunoprecipitated from both types of transduced cells (Fig. 1B and C, lane 3).


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)

Schematic map of the lentiviral vectors utilised in this study. (A) This vector is self-inactivating in transduced cells due to deletions of the enhancer region in the 3′ LTR. The reporter gene, GFP is driven by a housekeeping promoter PGK (top panel) and UCOE (bottom panel). (B) Flow cytometry analysis of porcine transduced spermatozoa. Porcine spermatozoa were incubated with a pSCF-VSV-g pseudotyped lentiviral vectors (Pgk promoter) and 7-AAD staining to exclude dead cells. (Top left panel) Mock/untransduced sperm light scatter analysis. (Top right panel) 7-AAD staining for exclusion of dead cells. (Bottom left panel) Untransduced (mock) live spermatozoa with no GFP expression. (Bottom right panel) Live and transduced spermatozoa showing 38% of spermatozoa expressing GFP.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

f0010: Schematic map of the lentiviral vectors utilised in this study. (A) This vector is self-inactivating in transduced cells due to deletions of the enhancer region in the 3′ LTR. The reporter gene, GFP is driven by a housekeeping promoter PGK (top panel) and UCOE (bottom panel). (B) Flow cytometry analysis of porcine transduced spermatozoa. Porcine spermatozoa were incubated with a pSCF-VSV-g pseudotyped lentiviral vectors (Pgk promoter) and 7-AAD staining to exclude dead cells. (Top left panel) Mock/untransduced sperm light scatter analysis. (Top right panel) 7-AAD staining for exclusion of dead cells. (Bottom left panel) Untransduced (mock) live spermatozoa with no GFP expression. (Bottom right panel) Live and transduced spermatozoa showing 38% of spermatozoa expressing GFP.
Mentions: Furthermore, when the VSV-g pseudotyped lentiviral vector encoding Histone 2B-fused Green Fluorescent Protein (H2B-GFP) (Fig. 2A top panel) was used to transduce 293T cells and porcine spermatozoa, newly synthesised GFP could be immunoprecipitated from both types of transduced cells (Fig. 1B and C, lane 3).

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