We previously reported that transgenic (TG) pigs can be produced from in vitro-matured oocytes using intracytoplasmic sperm injection-mediated gene transfer (ICSI-mediated method) (Kurome et al. 2006 Transgenic Res. 15, 229–240). We subsequently studied the expression of a foreign gene which had been introduced by the ICSI-mediated method. We found that the ICSI-mediated method is considerably less likely than the pronuclear microinjection method to produce embryos in which transgene-positive and transgene-negative cells co-exist, that is, mosaic embryos (Saito et al. 2006 Reprod. Fertil. Dev. 18, 297 abst). Therefore, in order to further investigate the ICSI-mediated method, the present study was conducted to address the integration patterns of foreign genes introduced by this method. In particular, we wished to determine the number of transgene copies and number of chromosomal integration sites. TG pig fetuses, obtained by the ICSI-mediated method in a separate cardiac disease model study, were used in the present study. Porcine cumulus-oocyte complexes that had been collected from slaughterhouse ovaries were subjected to in vitro maturation in NCSU23 medium to produce MII oocytes to be used in this study. Porcine spermatozoa frozen in Beltsville Thawing Solution (BTS) were thawed rapidly in a 37�C water bath, and each spermatozoon was decapitated using ultrasound (28 kHz, 100 W; W-113; Honda Electronics Co., Ltd, Aichi, Japan). The heads (2 to 5 � 105/10 �L) were co-incubated with 2.5 ng �L-1 of rabbit calreticulin cDNA (�MHC-CRT-HA: 7.5 kb) for five min at room temperature, and then microinjected into MII oocytes using a piezo-micromanipulator. An electric stimulus (DC 150 V mm-1, 100 �s) was applied 10 to 40 min after microinjection in order to activate the oocytes. The embryos were cultured in PZM-5 medium for one to two days, and then transferred into the oviducts of recipient gilts, whose estrous cycle had been synchronized using 1000 IU eCG and 1500 IU hCG. Fetuses were collected 33 or 50 days later, and a primary cell line (fibroblast) was established. For each fetus, the number of transgene copies was determined by Southern blot. In addition, the chromosomal sites, where the foreign gene had integrated, were identified, and the number of integration sites was determined by fluoresent in situ hybridization (FISH). A total of 454 ICSI embryos were transferred to 4 recipients (92 to 135 embryos/recipient). All recipients became pregnant and 23 fetuses (5.1%, 23/454), including 7 TG fetuses (30.4%, 7/23), were obtained. Southern blot analysis showed that the number of transgene copies varied between 1 and 300 (1 copy: 1 fetus; 10 copies: 2 fetuses; 30 copies: 3 fetuses; 300 copies: 1 fetus). FISH analysis showed that in TG fetuses, the foreign gene had integrated at only a single chromosomal site, and this site varied from TG fetus to TG fetus. These results demonstrate that, in the case of ICSI-mediated gene transfer, as is the case for gene transfer by pronuclear microinjection, the integration patterns are: multiple copy, random site, and single site integration.
This study was supported by PROBRAIN.
RAISING: a high-performance method for identifying random transgene integration sites
