Obtaining calves after transfer of embryos microin-jected with the human interferon alpha ( IFNα ) gene , pbLGIFN-GFPBsd

The objective of this study was to obtain calves with an integrated human interferon alpha (IFNα) gene. The transgene (pbLGIFN-GFPBsd) was introduced by microinjection into one of the zygote’s pronuclei. The transgene (pbLGIFN-GFPBsd) contained the human interferon alpha (IFNα) gene, bovine lactoglobulin promoter (bLG), gfp (green fluorescence protein) and blasticidin resistance gene (bsd). After microinjection of 2107 zygotes, the proportion developing to the blastocyst stage (10.4%) was lower than for non-injected embryos (39.9%) (P<0.01). Of the 2107 that were microinjected, 76 were GFP-positive blastocysts (3.6%). Fifty-seven GFP-positive blastocysts and 31 blastocysts from non-injected embryos were transferred singly to recipients. A lower rate of calving was observed after transfer of GFP-positive blastocysts (19.3%) as compared with noninjected embryos (48.4%) (P<0.05). All newborn calves were healthy and of normal weight. PCR analyses indicated that none of the calves obtained after transfer of GFP-positive blastocysts carried the human interferon alpha gene.


In vitro maturation of bovine oocytes
Bovine ovaries were obtained from a slaughterhouse and transported within 2 h to the laboratory in phosphate-buffered solution (PBS) with 100 IU/ml streptomycin and 100 IU/ml penicillin at approximately 30 o C. Cumulus-oocyte complexes (COCs) were collected by aspiration from follicles (2 to 6 mm in diameter) using a syringe with an 18-gauge needle and an air pump system.COCs were washed 3 times in manipulation medium (MM): TCM199 buffered with 25 mM Hepes supplemented with 10% FBS, 50 µg/ml gentamicin sulphate, 100 IU penicillin and 50 µg/ml streptomycin, and adjusted to pH 7.4.A group of COCs (20 immature oocytes) was placed in one well of a 4-well dish and matured in 450 µl TCM199 l TCM199 TCM199 buffered with 25 mM Hepes and supplemented with 10% FBS, 0.02 IU/ml pFSH, 1 µg/ml 17 �-estradiol, 0.2 mM Na pyruvate and 50 µ/ml gentamicin sulphate, g/ml 17 �-estradiol, 0.2 mM Na pyruvate and 50 µ/ml gentamicin sulphate, ml 17 �-estradiol, 0.2 mM Na pyruvate and 50 µ/ml gentamicin sulphate, and adjusted to pH 7.4.The COCs were matured for 24 h at 38.5 o C in 5% CO 2 in humidified air.

In vitro fertilization of bovine oocytes
Frozen spermatozoa obtained from a single bull were thawed in a water bath (37 o C), centrifuged (200 g) for 10 min, and resuspended in 2 ml Sp-TALP medium containing 6 mg/ml BSA fraction V adjusted to pH 7.4.Spermatozoa were prepared by the swim-up procedure.After maturation, the COCs were washed 3 times in fertilization medium Fert-TALP supplemented with 6 mg/ml BSA FAF, 0.2 mM Na pyruvate and 50 µg/ml gentamicin sulphate.Groups of matured oocytes (10 COCs) were placed in 4-well dishes in 450 µl Fert-TALP supplemented with 6 mg/ l Fert-TALP supplemented with 6 mg/ Fert-TALP supplemented with 6 mg/ ml BSA FAF, 0.2 mM Na pyruvate, 50 µg/ml gentamicin sulphate, 20 µM penicillamine, 10 µM hypotaurine, 1 µM epinephrine and 2 µg/ml heparin.Spermatozoa were used at a final concentration of 1x10 6 /ml.The COCs and spermatozoa were co-cultured for 18 h at 38.5 o C in 5% CO 2 in humidified air.

Microinjection of the pbLGIFN-GFPBsd construct into bovine zygotes
At 18 h post-insemination the cumulus cells were removed and the zygotes were washed in manipulation medium (MM).Zygotes were transferred to a microcentrifuge tube into 50 µl of MM and centrifuged for 6 min at 12000 g at room temperature.They were then transferred to a manipulation chamber (15 µl drops of MM, covered with mineral oil).The manipulation chamber was placed under an Inverted Microscope with Nomarski contrast optics and micromanipulator (Leitz).The pbL-GIFN-GFPBsd construct was diluted to 3 ng/�l in TE buffer (pH 8.0).Microinjec-�l in TE buffer (pH 8.0).Microinjec-l in TE buffer (pH 8.0).Microinjections into one of the pronuclei were performed and the times of injections were noted by observing the swelling of the pronucleus.Microinjections were performed between 18-20 h post-insemination.

In vitro culture of bovine embryos
Non-injected and microinjected zygotes were washed in Menezo B2 medium (ART of CCD) supplemented with 10% FBS (GIBCO) and placed in 40 µl drops of Menezo B2 medium supplemented with 10% FBS under mineral oil.Zygotes from the microinjected group and non-injected group were co-cultured on Vero cell monolayers (ATCC, Maryland, USA).Vero cells at a concentration of 2�10 (ATCC, Maryland, USA).Vero cells at a concentration of 2�10 Vero cells at a concentration of 2�10 3 /10 µl were placed into a microdrop (40 µl) of Menezo B2 (Assisted Reproductive Technology of Laboratoire CCD, France) supplemented with 10% FBS under mineral oil.Zygotes were cultured until day 7 (168 h post-insemination), at 38.5°C in 5% CO 2 in a humidified incubator.During culture, the medium was renewed three times (at 48 and 72 h 20 µl of medium were removed and 20 µl of Menezo B2 supplemented with 10% FBS were added; at 144 h, 20 µl of medium were removed and 20 µl of Menezo B2 without serum were added).Development of the embryos was evaluated at 48 Development of the embryos was evaluated at 48 and 168 h post-insemination.

Detection of GFP expression by fluorescence analysis
At 48 h post-insemination, 357 embryos from the microinjected group and at 168 h post-insemination, 218 embryos from the microinjected group and 10 embryos from the non-injected group were examined individually using a Fluovert FS microscope (Leitz) with excitation of the chromophore at 488 nm and a standard GFP filter.The embryos were scored for GFP-positive blastomeres within each of them.A GFP-positive embryo was one in which at least one blastomere yielded green fluorescence.

Embryo transfer and offspring evaluation
The control and GFP-positive blastocysts were transferred to recipients.Oestrous synchronization of the recipients was induced by injecting 2 ml (0.5 mg) of the prostaglandin F 2α -analogue Cloprostenol (Bioestrovet-Vetoquinol, Gorzów, Poland) every 11 days.Seven to eight days after standing heat, one fresh embryo was transferred to the uterine horn ipsilaterally to the ovary displaying a corpus luteum.Embryos were transferred to recipients in Embryo Transfer Medium (BioLife Transfer Medium, Agtech Inc., USA).Recipients were monitored daily for heat behaviour and examined by ultrasound after 5 weeks, and then monthly, to confirm pregnancy.After calving, offspring were weighed and subjected to veterinary examination.
PCR analyses of genomic DNA isolated from blood and ear cells were carried out for all newborn calves from the microinjected group and the non-injected group.

Statistical analysis
Rates of cleavage and blastocyst formation per zygote and rate of development after transfer to recipients were analysed by Chi-square tests.Probabilities (P-values) of less than 0.05 were considered statistically significant and less than 0.01 highly statistically significant.
PCR analyses indicated that none of the calves obtained after transfer of GFPpositive embryos carried the pbLGIFN-GFPBsd transgene.
Only one microinjected embryo was 100% GFP-positive and the others were mosaic embryos.The mosaicism in embryos is connected with the process of integration into the host genome.This process is time-limited and the frequency of integration would be expected to decrease with each successive cell division, which contributes to mosaicism.The non-integrated DNA can be degraded over time (Chan et al., 1999).
All newborn calves from the microinjected group were obtained after transfer of mosaic embryos to recipients.Resorption was observed in the case of the 100% GFP-positive embryo.None of the calves obtained after transfer of GFP-positive embryos showed the presence of the human interferon alpha (IFNα) gene in either their blood or ear tissues, as tested by PCR.Perhaps the introduced transgene was integrated into other tissues which were not analysed.

CONCLUSIONS
Although the calves were born after transfer of pbLGIFN-GFPBsd microinjected embryos, none of them carried the human interferon alpha transgene.

Table 1 .
In vitro development of microinjected and non-injected bovine embryos

Table 2 .
Results for the transfer of GFP positive and non-injected embryos to recipients