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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 vector detection and integration of vectors into porcine spermatozoon genome. (A) Detection of WPRE sequence from lentivirally transduced spermatozoa by PCR of sperm genomic DNA. Lanes 1: 100 bp Molecular weight marker-Promega, 2: water control (no template DNA), 3: Genomic DNA from 293T cells stably transduced lentiviral vectors, 4: Genomic DNA from porcine sperm transduced with lentiviral vectors (VSV-g only) for 48 h, 5: Genomic DNA from porcine sperm transduced with lentiviral vectors (pSCF-VSV-g) for 48 h, 6: Mock (untransduced control) sperm DNA. (B) Following LAM-PCR, amplified products were cloned and sequenced. The first unique sequence, integrated to the x-chromosome of the porcine genome. The alignments shown were from two colonies containing similar integration sites.
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f0025: Lentiviral vector detection and integration of vectors into porcine spermatozoon genome. (A) Detection of WPRE sequence from lentivirally transduced spermatozoa by PCR of sperm genomic DNA. Lanes 1: 100 bp Molecular weight marker-Promega, 2: water control (no template DNA), 3: Genomic DNA from 293T cells stably transduced lentiviral vectors, 4: Genomic DNA from porcine sperm transduced with lentiviral vectors (VSV-g only) for 48 h, 5: Genomic DNA from porcine sperm transduced with lentiviral vectors (pSCF-VSV-g) for 48 h, 6: Mock (untransduced control) sperm DNA. (B) Following LAM-PCR, amplified products were cloned and sequenced. The first unique sequence, integrated to the x-chromosome of the porcine genome. The alignments shown were from two colonies containing similar integration sites.

Mentions: Lentivirally transduced porcine spermatozoa were further analysed for the presence of the lentiviral vector genome in sperm. This was done by PCR amplification of the WPRE sequence encoded within the lentiviral vector genome from lentivirally transduced porcine sperm genomic DNA. The expected 174 bp PCR fragment was successfully amplified (Fig. 5A).


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 vector detection and integration of vectors into porcine spermatozoon genome. (A) Detection of WPRE sequence from lentivirally transduced spermatozoa by PCR of sperm genomic DNA. Lanes 1: 100 bp Molecular weight marker-Promega, 2: water control (no template DNA), 3: Genomic DNA from 293T cells stably transduced lentiviral vectors, 4: Genomic DNA from porcine sperm transduced with lentiviral vectors (VSV-g only) for 48 h, 5: Genomic DNA from porcine sperm transduced with lentiviral vectors (pSCF-VSV-g) for 48 h, 6: Mock (untransduced control) sperm DNA. (B) Following LAM-PCR, amplified products were cloned and sequenced. The first unique sequence, integrated to the x-chromosome of the porcine genome. The alignments shown were from two colonies containing similar integration sites.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

f0025: Lentiviral vector detection and integration of vectors into porcine spermatozoon genome. (A) Detection of WPRE sequence from lentivirally transduced spermatozoa by PCR of sperm genomic DNA. Lanes 1: 100 bp Molecular weight marker-Promega, 2: water control (no template DNA), 3: Genomic DNA from 293T cells stably transduced lentiviral vectors, 4: Genomic DNA from porcine sperm transduced with lentiviral vectors (VSV-g only) for 48 h, 5: Genomic DNA from porcine sperm transduced with lentiviral vectors (pSCF-VSV-g) for 48 h, 6: Mock (untransduced control) sperm DNA. (B) Following LAM-PCR, amplified products were cloned and sequenced. The first unique sequence, integrated to the x-chromosome of the porcine genome. The alignments shown were from two colonies containing similar integration sites.
Mentions: Lentivirally transduced porcine spermatozoa were further analysed for the presence of the lentiviral vector genome in sperm. This was done by PCR amplification of the WPRE sequence encoded within the lentiviral vector genome from lentivirally transduced porcine sperm genomic DNA. The expected 174 bp PCR fragment was successfully amplified (Fig. 5A).

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