title: Sperm status and DNA dose play key roles in sperm/ICSI-mediated gene transfer in caprine.
abstract: In relation to the growing recent interest in the establishment of sperm-mediated gene transfer (SMGT) technology as a convenient and effective method for the simple production of transgenic animals, in this study the possibility of using SMGT to produce transgenic caprine embryos was investigated for the first time. Buck sperm were directly incubated with different concentrations (0-500 ng) of pcDNA/his/Lac-Z plasmid and used for IVF or ICSI. Sperm used for ICSI were categorized into motile or live-immotile group before being injected into oocytes. In a separate experiment, dead sperm prepared by repeated freezing/thawing were used for DNA-incubation before ICSI. Sham injection was carried out by intracytoplasmic injection of approximately the same volume of media containing different doses of DNA using an ICSI needle. Transgene expression and transmission were detected by X-Gal staining and PCR analysis of developed embryos, respectively. A reasonable blastocyst rate was observed in all the groups. Only embryos in the sham group were negative for transgene transmission. Transgene expression was completely dependent on the delivery technique and status of sperm, and was only observed in the live-immotile and dead ICSI groups. The results of this study showed that the technique (IVF vs. ICSI vs. sham injection), sperm status (motile vs. live-immotile vs. dead) and to some extent DNA concentration affect embryo development, transgene transmission and expression.
authors.affiliation: Department of Reproduction and Development, Royan Institute for Animal Biotechnology, ACECR, Isfahan, Iran.
authors.forename: Farshad
authors.initials: F
authors.lastname: Shadanloo
authors.forename: Mohammad Hasan
authors.initials: MH
authors.lastname: Najafi
authors.forename: Sayed Morteza
authors.initials: SM
authors.lastname: Hosseini
authors.forename: Mehdi
authors.initials: M
authors.lastname: Hajian
authors.forename: Mohsen
authors.initials: M
authors.lastname: Forouzanfar
authors.forename: Kamran
authors.initials: K
authors.lastname: Ghaedi
authors.forename: Parvaneh
authors.initials: P
authors.lastname: Abedi
authors.forename: Somaye
authors.initials: S
authors.lastname: Ostadhosseini
authors.forename: Laleh
authors.initials: L
authors.lastname: Hosseini
authors.forename: Morad-Pasha
authors.initials: MP
authors.lastname: Eskandari-Nasab
authors.forename: Mohammad Hossein Nasr
authors.initials: MH
authors.lastname: Esfahani
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D004247:DNA / Q000008:administration & dosage / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004622:Embryo, Mammalian / Q000378:metabolism; D005260:Female; D018014:Gene Transfer Techniques; D006041:Goats / Q000196:embryology*; D016015:Logistic Models; D008297:Male; D009865:Oocytes / Q000378:metabolism; D016133:Polymerase Chain Reaction; D020554:Sperm Injections, Intracytoplasmic / Q000379:methods*; D013094:Spermatozoa / Q000737:chemistry / Q000502:physiology
chemical_list: D004247:DNA