Lentiviral vector-mediated transduction of goat undifferentiated spermatogonia.
Recent studies show that spermatogonial stem cells (SSCs) are able to colonize and form mature spermatozoa following transplantation into germ cell depleted testes of recipient males. Therefore, efficient ways for enrichment and gene transfer into SSCs provides a powerful tool for production of transgenic animals. In order to adapt the technique to goats, three issues were addressed: (i) enrichment of the undifferentiated spermatogonia including SSCs using magnetic activated cell sorting (MACS), (ii) lentiviral vector-mediated transduction of an enhanced green fluorescent protein (EGFP) transgene into enriched cells, and (iii) transplantation of transduced undifferentiated spermatogonia into the germ cell depleted testes of immune-suppressed mice to assess for migration and colony formation ability. Enriched cells were transduced by lentiviral vectors and subsequently analyzed for expression of THY1, PLZF, VASA, UCHL1 and BCL6B genes. Cells were also analyzed for GFP and PLZF by flow cytometry. Enriched transduced cells were transplanted into germ cell depleted mice testis. Quantitative analysis of transcripts revealed that MACS-enrichment significantly increased the expression of SSC-characteristic genes THY1, PLZF, VASA, UCHL1 and BCL6B compared to non-enriched population (P≤0.05). EGFP transduction did not affect the expression levels of SSC-characteristic genes. Flow cytometry revealed that 72% of transduced-enriched cells were positive for EGFP. Finally, transduced-enriched goat SSCs could colonize within the cells into the seminiferous tubules of germ cell depleted recipient mice at higher frequency than non-enriched cells. The results indicated that enrichment of goat undifferentiated spermatogonia by magnetic-activated cell sorting for THY1 antibody combined with lentiviral vector-mediated transduction has the potential to be used for production of transgenic goats.
Department of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad, Iran.
Hassan
H
Abbasi
Department of Reproductive Biotechnology at Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran.
Sayyed Morteza
SM
Hosseini
Department of Reproductive Biotechnology at Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran.
Mahdi
M
Hajian
Department of Reproductive Biotechnology at Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran.
Zahra
Z
Nasiri
Department of Biology, Falavarjan Branch, Islamic Azad University, Isfahan, Iran.
Mehrnoosh
M
Bahadorani
Department of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad, Iran.
Mojtaba
M
Tahmoorespur
Department of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad, Iran.
Mohammad Reza
MR
Nasiri
Department of Reproductive Biotechnology at Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran; Department of Cellular Biotechnology at Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran. Electronic address: mh.nasr-esfahani@royaninstitute.org.
Mohammad Hossein
MH
Nasr-Esfahani
D000818:Animals; D000906:Antibodies; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D006041:Goats; D049452:Green Fluorescent Proteins; D008297:Male; D051379:Mice; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012671:Seminiferous Tubules; D013093:Spermatogonia / Q000637:transplantation*; D013234:Stem Cells / Q000502:physiology*; D018800:Thy-1 Antigens / Q000276:immunology
D000906:Antibodies; D018800:Thy-1 Antigens; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
Goat; Lentiviral vectors; MACS; SSCs; THY1; Transduction