Coupling sperm mediated gene transfer and sperm sorting techniques: a new perspective for swine transgenesis.
Flow cytometric separation of X and Y chromosome-bearing spermatozoa has been demonstrated to be effective in pigs, allowing the use of boar sexed semen in in vitro trials. Sperm Mediated Gene Transfer (SMGT) is a widely used and efficient technique for the creation of transgenic animals. The present research intended to prove that it is possible to associate sperm sexing with the SMGT technique in order to speed up the assessment of homozygous lines of transgenic pigs. In the first experiment, the sorting protocol was modified in order to obtain the highest DNA uptake by sorted spermatozoa. In the second experiment, spermatozoa that had undergone only sperm sorting, only SMGT, or both procedures (Sorted-SMGT) were used for in in vitro fertilization of in vitro matured oocytes. In the third experiment, transformed blastocysts of the desired gender (male) were obtained with Sorted-SMGT in an in vitro fertilization trial. The method we developed here allowed us to produce transgenic swine blastocysts of pre-determined gender, giving a positive answer at the aim to couple SMGT and sperm sorting in swine, obtaining fertile spermatozoa able to produce transgenic embryos of pre-determined gender.
De Cecco
Marco
M
Department of Veterinary Morphophysiology and Animal Production-DIMORFIPA, University of Bologna, Bologna, Italy.
Spinaci
Marcella
M
Zannoni
Augusta
A
Bernardini
Chiara
C
Seren
Eraldo
E
Forni
Monica
M
Bacci
Maria Laura
ML
D000818:Animals; D030801:Animals, Genetically Modified; D001755:Blastocyst / Q000502:physiology; D046149:Embryo Culture Techniques; D004622:Embryo, Mammalian; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary; D005434:Flow Cytometry / Q000379:methods / Q000662:veterinary; D018014:Gene Transfer Techniques / Q000662:veterinary*; D005818:Genetic Engineering / Q000379:methods / Q000662:veterinary*; D008297:Male; D012743:Sex Preselection / Q000662:veterinary*; D013094:Spermatozoa / Q000166:cytology; D013552:Swine / Q000196:embryology / Q000235:genetics*