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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

De novo protein synthesis and GFP precipitation from vector transduced spermatozoa. (A) Newly synthesised proteins could be detected following culture of porcine sperm with radioactively labelled methionine. Gel blots were dried to completion onto filter paper, exposed to an X-ray film for 2 weeks and X-ray film developed using an automatic developer. (B) 293T cells were incubated with VSV-g pseudotyped lentivectors (with histone 2b-gfp as the reporter) at MOIs ranging from 1–100 in the presence of 35S labelled methionine for 4 h, GFP could be immunoprecipitated quantitatively with an anti GFP antibody. On the other hand, 293T cells that had been transduced with same vector and propagated for 3 weeks in culture and then subjected to radiolabeling (4 h) and immunoprecipitation with antibody to GFP also revealed the 48 kDa histone2b-gfp marker but did not differ quantitatively. (C) In spermatozoa, GFP could also be immunoprecipitated as seen in lane 3. Mock transduced and protein G beads only (no antibody) controls are in lanes 1 and 2 respectively. Total 35S radiolabelled proteins (from mock, agarose-beads only and vector incubated lysates) prior to immunoprecipitation are represented in lanes 5–7 respectively. Exposure of blots to an auto radiography film was carried out for 2 weeks.
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f0005: De novo protein synthesis and GFP precipitation from vector transduced spermatozoa. (A) Newly synthesised proteins could be detected following culture of porcine sperm with radioactively labelled methionine. Gel blots were dried to completion onto filter paper, exposed to an X-ray film for 2 weeks and X-ray film developed using an automatic developer. (B) 293T cells were incubated with VSV-g pseudotyped lentivectors (with histone 2b-gfp as the reporter) at MOIs ranging from 1–100 in the presence of 35S labelled methionine for 4 h, GFP could be immunoprecipitated quantitatively with an anti GFP antibody. On the other hand, 293T cells that had been transduced with same vector and propagated for 3 weeks in culture and then subjected to radiolabeling (4 h) and immunoprecipitation with antibody to GFP also revealed the 48 kDa histone2b-gfp marker but did not differ quantitatively. (C) In spermatozoa, GFP could also be immunoprecipitated as seen in lane 3. Mock transduced and protein G beads only (no antibody) controls are in lanes 1 and 2 respectively. Total 35S radiolabelled proteins (from mock, agarose-beads only and vector incubated lysates) prior to immunoprecipitation are represented in lanes 5–7 respectively. Exposure of blots to an auto radiography film was carried out for 2 weeks.

Mentions: To establish that mature spermatozoa are capable of de novo protein synthesis we performed radioisotope labelling of actively synthesised proteins and immunoprecipitation studies. When pig sperm were cultured in methionine-free medium supplemented with 35S labelled methionine, under conditions favouring capacitation [20], newly synthesised proteins could be detected by autoradiography following polyacrylamide gel electrophoresis (Fig. 1A). This observation was consistent with previous findings that mature spermatozoa are thought to translate nuclear encoded genes by mitochondrial type ribosomes contained in sperm [20].


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)

De novo protein synthesis and GFP precipitation from vector transduced spermatozoa. (A) Newly synthesised proteins could be detected following culture of porcine sperm with radioactively labelled methionine. Gel blots were dried to completion onto filter paper, exposed to an X-ray film for 2 weeks and X-ray film developed using an automatic developer. (B) 293T cells were incubated with VSV-g pseudotyped lentivectors (with histone 2b-gfp as the reporter) at MOIs ranging from 1–100 in the presence of 35S labelled methionine for 4 h, GFP could be immunoprecipitated quantitatively with an anti GFP antibody. On the other hand, 293T cells that had been transduced with same vector and propagated for 3 weeks in culture and then subjected to radiolabeling (4 h) and immunoprecipitation with antibody to GFP also revealed the 48 kDa histone2b-gfp marker but did not differ quantitatively. (C) In spermatozoa, GFP could also be immunoprecipitated as seen in lane 3. Mock transduced and protein G beads only (no antibody) controls are in lanes 1 and 2 respectively. Total 35S radiolabelled proteins (from mock, agarose-beads only and vector incubated lysates) prior to immunoprecipitation are represented in lanes 5–7 respectively. Exposure of blots to an auto radiography film was carried out for 2 weeks.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

f0005: De novo protein synthesis and GFP precipitation from vector transduced spermatozoa. (A) Newly synthesised proteins could be detected following culture of porcine sperm with radioactively labelled methionine. Gel blots were dried to completion onto filter paper, exposed to an X-ray film for 2 weeks and X-ray film developed using an automatic developer. (B) 293T cells were incubated with VSV-g pseudotyped lentivectors (with histone 2b-gfp as the reporter) at MOIs ranging from 1–100 in the presence of 35S labelled methionine for 4 h, GFP could be immunoprecipitated quantitatively with an anti GFP antibody. On the other hand, 293T cells that had been transduced with same vector and propagated for 3 weeks in culture and then subjected to radiolabeling (4 h) and immunoprecipitation with antibody to GFP also revealed the 48 kDa histone2b-gfp marker but did not differ quantitatively. (C) In spermatozoa, GFP could also be immunoprecipitated as seen in lane 3. Mock transduced and protein G beads only (no antibody) controls are in lanes 1 and 2 respectively. Total 35S radiolabelled proteins (from mock, agarose-beads only and vector incubated lysates) prior to immunoprecipitation are represented in lanes 5–7 respectively. Exposure of blots to an auto radiography film was carried out for 2 weeks.
Mentions: To establish that mature spermatozoa are capable of de novo protein synthesis we performed radioisotope labelling of actively synthesised proteins and immunoprecipitation studies. When pig sperm were cultured in methionine-free medium supplemented with 35S labelled methionine, under conditions favouring capacitation [20], newly synthesised proteins could be detected by autoradiography following polyacrylamide gel electrophoresis (Fig. 1A). This observation was consistent with previous findings that mature spermatozoa are thought to translate nuclear encoded genes by mitochondrial type ribosomes contained in sperm [20].

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