. mg from each calf was digested with DraI, EcoRI or HindIII and then separated on a
0.7 agarose gel. After transfer to a nylon membrane, the DNA was hybridized with a
32
P-labeled DNA probe for 24 h, rinsed with 0.1 = SSCr0.1 SDS solution and then exposed to an X-ray film. The DNA used for the probe was the 2.0 kb SmaI–EcoRI
Ž .
fragments of human lactoferrin cDNA Kim et al., 1994 . 2.9. Statistical analysis
The pregnancy rate after transfer of DNA-injected bovine embryos was analyzed by the Student’s t-test. The survival rate of the DNA-injected bovine embryos after freezing
and thawing was analyzed by the Duncan’s multiple range test using the general linear models in the statistical analysis system. Survival rates of DNA-injected bovine blasto-
cysts after freezing and thawing according to embryo quality and developmental stage are described as mean SD. All percentage data were subjected to arcsine transforma-
tion. Probability of P - 0.05 was considered to be statistically significant.
3. Results
The overall survival rate of DNA-injected bovine blastocysts after freezing and Ž
. thawing was 59 72r123 . Fig. 1A shows that the survival rate of DNA-injected
Ž .
blastocysts with excellent quality after freezing and thawing 78.8 10.6, 37r46 was
Ž . Ž .
Fig. 1. Effects of embryo quality A and developmental stage B on the survival rate of DNA-injected bovine embryos after freezing and thawing. Differences were significantly detected in the survival rate of the embryos
Ž .
after freezing and thawing between groups of embryo quality or developmental stage P - 0.05 . EX,
expanded blastocyst; MB, mid-blastocysts; EB, early blastocyst.
Ž .
Ž .
Ž higher than those of good P - 0.05 and fair P - 0.05 quality 60.9 16.4, 32r53
. and 12.5 5.9, 3r24, respectively . The difference between good and fair was
Ž .
significant P - 0.05 . Survival rates of DNA-injected bovine blastocysts at different
stages of development when frozen are shown in Fig. 1B. Expanded blastocysts Ž
. Ž
71.2 1.1, 35r49 showed higher survival rate than those of early 52.1 12.6, .
Ž .
Ž .
31r61 and mid- 48.8 15.9, 6r13 blastocysts P - 0.05 . After IVM and IVF, 10 252 zygotes were injected with pBL1 and cultured in CR1aa
Ž medium supplemented with 4 mgrml BSA for 48 h. When cleaved embryos 52.6,
. 5391r10 252 were further co-cultured with MEF for 5 days, the developmental rate to
Ž .
blastocyst stage was 13.4 722r5391 . Transfer of the frozen–thawed DNA-injected Ž
. bovine embryos resulted in a pregnancy rate of 13.6 13r96, Table 1 . This pregnancy
Ž .
Ž .
rate was lower than that 26.5, 43r162 of transferred fresh embryos P - 0.05 . Most Ž
. pregnant recipients transferred with frozen embryos delivered normal live calves 12r13
and one calf was still-born. In the fresh embryo transfer group, the number of abortions and still-births was 9r43. In this study, no histopathological tests were not performed on
the still-born calves. A preponderance of male calves was produced from transfer of
Ž .
Ž .
both fresh M:F 9:3 and frozenrthawed M:F 20:14 embryos. Two calves derived from DNA-injected embryos after freezing and thawing were identified as transgenic by
Southern blot analysis, although no transgenic animals were detected in the calves resulting from fresh embryo transfers. Both the transgenic calves were males and
appeared to be normal. Fig. 2 shows the Southern blot of the first transgenic calf. The
Ž .
transgene bovine b-caseinrhuman lactoferrin cDNA fusion gene has only one site for Ž
. Ž
a restriction enzyme HindIII. Genomic DNAs 10 mg were obtained from blood lanes .
Ž .
3, 4, 7, 8, 9,12, 13, 14 and 17 and ear tissues lanes 5, 6, 10, 11, 15 and 16 of three Ž
. calves a3626, a3629 and a3753 , and then digested with restriction enzymes of DraI
Ž .
Ž .
lanes 1, 3, 5, 8, 10, 13, 15 and 18 , EcoRI lanes 2, 4, 6, 9, 11, 14, 16 and 19 and Ž
. HindIII lanes 5, 10 and 15 . After electrophoresis and transfer to a nylon membrane, the
DNA samples were probed with
32
P-labeled fragments of human lactoferrin cDNA. Ž
. Ž
Transgene was identified in both the blood lanes 8, 9 and 12 and ear samples lanes 10 .
Ž .
and 11 of one calf a3628 . The density ratio of the upper and lower bands was Ž
. approximately 1:3 determined by Phosphoimazer . This result shows that the founder
transgenic bull has four copies of the transgene as a concatamer. The second transgenic Ž
. calf had one copy of the transgene. To date, 3 out of 23 calves 13.0 born from cows
Table 1 Production of transgenic calves after the transfer of DNA-injected bovine embryos
Group No. of embryos
Pregnant recipients Live-born
Transgenic calves
a b
c
Ž . Ž
. Ž .
transferred MrF
Ž .
Ž .
Ž .
Frozen 153
13r96 13.6 12 9r3
2 18.3 Ž
. Ž
. Fresh
242 43r162 26.5
34 20r14
a
Pregnant at 60 days of gestation.
b
M s male, F s female.
c
Transgenic calvesrlive-born calves=100. P - 0.05.
Fig. 2. Identification of a transgenic calf by Southern blot analysis. The sizes of DNA marker, named as l r HindIII DNA fragments applied on lane M, were as follows; 2.0, 2.3, 4.3, 6.5, 9.4 and 23.1 kbp from
bottom to top. Lanes 1 and 2 are 5 pg of injected DNA fragment digested with DraI and EcoRI, respectively, as positive controls. Lanes 18 and 19 are 10 mg of non-transgenic genomic DNAs digested with DraI and
EcoRI, respectively, as negative controls.
inseminated with the semen of the transgenic bull were transgenic. Thus, mosaicism of the founder bull was demonstrated. It was shown by PCR and Southern blot analyses
Ž .
that the second transgenic bull had no transgenic spermatozoa data not shown .
4. Discussion
