ined whether transgenic bulls derived from frozenrthawed embryos pass the transgenes to their progeny through the germ-line.
2. Materials and methods
2.1. IVM and IVF Ž
. IVM and IVF of bovine oocytes were performed as described by Han et al. 1996 .
Briefly, 10–20 immature oocytes obtained from slaughtered Holstein cows were cul- Ž
tured in 0.5 ml of maturation medium containing 1 mgrml estradiol Sigma, St Louis, .
Ž .
MO and 1 mgrml FSH-Pe Schering-Plough Animal Health, Kenilworth, NJ for 22–24 h at 38.58C, 5 CO
in humidified air. The maturation medium consisted of
2
Ž .
TCM-199 with Eagle’s salts and
L
-glutamine supplemented with 10 vrv heat-in- Ž
. activated fetal bovine serum FBS; Gibco BRL and 25 mM NaHCO . After IVM, 10
3
oocytes were fertilized with frozen–thawed sperm at a concentration of 2 = 10
6
rml in Ž
. 50 ml of fertilization medium Bavister and Yanagimachi, 1977 . When sperm were
added to the fertilization drops, 2 mgrml heparin, 20 mM penicillamine, 10 mM Ž
. hypotaurine and 1 mM epinephrine PHE were also added. After 18 h of insemination,
cumulus-enclosed oocytes were stripped by vortexing for 2 min and placed in CR1aa medium before DNA injection. CR1aa medium was formulated according to the
Ž .
procedures of Rosenkrans et al. 1993 , and supplemented with 1 mM glutamine and Ž
. 1 = Eagle’s essential amino acids solution Gibco BRL .
2.2. Microinjection of DNA The DNA used for microinjection was 5.5 kbp fragments of an expression vector,
pBL1, which consisted of the bovine b-casein gene promoter, human lactoferrin cDNA Ž
. and SV40 polyadenylation signal Kim et al., 1994 . The DNA concentration was
adjusted to 4 mgrml in a buffer of 10 mM Tris–HCl, pH 8.0, and 0.1 mM EDTA. The microinjection technique was similar to the methods previously described for pig and
Ž .
sheep embryos Hammer et al., 1985 . In order to visualize the pronuclei, cumulus-free oocytes were centrifuged in 1 ml TL–Hepes medium at 15 000 = g for 7 min in a
Ž .
microcentrifuge Vision Scientific, Korea . The embryos were then injected into one pronucleus with a DNA solution at 21–25 h after insemination. Swelling of the
pronucleus indicated that the injection had been successful. After DNA injection, groups of 5–10 surviving zygotes were cultured for 2 days in 50 ml drops of CR1aa medium
Ž .
supplemented with 3 mgrml BSA under light mineral oil Sigma . 2.3. Mouse embryonic fibroblasts MEF monolayer
Ž .
MEF monolayers were prepared as described by Joyner 1993 . Briefly, individual fetus was collected aseptically at necropsy from pregnant mice from days 14 to 16, and
then finely minced with microscissors. The minced tissues were washed three times with PBS free of Ca
2q
and Mg
2q
ions, and trypsinized in 0.1 trypsinr0.05 EDTA solution while being stirred. Suspended cells were sieved through a stainless mesh
Ž .
Ikemoto, Tokyo, Japan and centrifuged at 700 = g for 5 min. The cell pellet from each Ž
. fetus was suspended in DMEM
Gibco BRL containing 10 FBS and the cell
suspension was placed in a 100 mm petri dish. The primary fibroblasts were cultured for 2 days to confluency at 378C, 5 CO
in humidified air, proliferated through two
2
subsequent passages as described above and then frozen at y708C. The freezing Ž
. Ž
. medium for the fibroblasts was 10 vrv glycerol plus 50 vrv FBS in PBS.
Ž .
Frozen MEF were thawed in 378C water and then grown in petri dishes 100 mm for 2 Ž
. days to confluency. MEF were treated with 10 mgrml mitomycin C Sigma to cause
inactivation for 2.5 h and then harvested. To co-culture bovine embryos, mitomycin C-treated cells were rinsed three times with PBS, suspended in DMEM containing 10
FBS and then plated at a concentration of 6 = 10
5
cellsrml in a four-well dish to be a monolayer. Approximately 1 h later, almost all DMEM was removed and replaced with
0.5 ml of CR1aa medium containing 10 FBS at least 2 h before use.
2.4. IVC Ž
. Ten to 15 cleaved four- to eight-cell embryos were selected at approximately 48 h
after DNA injection and then further co-cultured with MEF for 5 days in 0.5 ml of Ž
. CR1aa medium supplemented with 10 vrv FBS at 38.58C, 5 CO in humidified
2
air. At day 5 of culture, the culture medium was supplemented with 5.56 mM glucose Ž
. and 1 = GMS-X supplement Gibco BRL without medium change. GMS-X supplement
solution consisted of 1.0 mgrml insulin, 0.67 mgrml sodium selenite, 0.55 mgrml transferrin and 0.2 mgrml ethanolamine. The day of in vitro fertilization was designated
as day 0 of culture.
2.5. Freezing and thawing of embryos The blastocysts that developed from DNA-injected bovine embryos were separately
Ž .
washed in PBS supplemented with 15 vrv FBS and then frozen by a modified Ž
. conventional slow freezing method Massip et al., 1987 . Briefly, the embryos were
Ž .
equilibrated for 10 min in freezing medium, consisting of 10 vrv glycerol and 50 FBS in PBS at room temperature and then loaded into 0.25 ml straws with a maximum
Ž of five embryos per straw. The straws were sealed with straw powder FHK, Fujihira
. Industry, Japan and placed at y58C in the cooling chamber of an automatic cell freezer
Ž .
SACO ACF-101: San Cheon Tech-Ind, Korea . After 2 min at y58C, seeding was induced, and 10 min later, cooling was initiated at 0.58Crmin to y308C followed by
plunging and storing the straws into liquid nitrogen. Thawing was conducted by agitating each straw in water at 378C for 20 s. The contents of each straw were expelled
into a petri dish. Embryos were rehydrated in stepwise diluents of 6, 3 and 0 glycerol for 5 min in each step. The diluent was based on PBS supplemented with 0.25
M sucrose and 0.5 BSA. The embryos were stepwise washed in two fresh solutions of PBS containing 30 and 15 FBS, respectively, and then co-cultured for at least 12 h
Ž .
in CR1aa supplemented with 10 vrv FBS on MEF monolayers at 38.58C, 5 CO
2
in air prior to embryo transfer. Survival of frozen–thawed bovine embryos were evaluated in terms of their morphological recovery and re-expansion of blastocysts after
approximately 12 h.
2.6. Embryo transfer Experiments were conducted according to The Animal Care and Use Committee
guidelines of the Korean Research Institute of Bioscience and Biotechnology. DNA-in- jected blastocysts were transferred to recipients by a non-surgical method. All recipients
were virgin Holstein heifers, 14 to 18 months old. One or two blastocysts, depending on the availability of embryos each day, were transferred to the unilateral uterine horn of a
recipient heifer on day 7 or 8 after a spontaneous estrus. Following transfer, pregnancy was confirmed by rectal palpation at approximately 60 days of gestation. The pregnant
recipients were normally managed to deliver their calves at the term.
2.7. Experimental designs 2.7.1. Experiment 1
It was investigated whether in vitro viability of DNA-injected bovine embryos produced in vitro was affected by freezing. All blastocysts that developed from
Ž DNA-injected embryos were individually classified into three grades excellent, good
. Ž
and fair embryos and different developmental stages early, mid and expanded blasto- .
Ž .
cysts by morphological appearance Lindner and Wright, 1983; Han et al., 1994 . Embryos judged with the same quality and developmental stage were loaded into a straw
and then frozen by a modified conventional slow freezing method. Following thawing, the survival of frozen embryos was evaluated in terms of their morphological recovery
and re-expansion of blastocysts after approximately 12 h of culture.
2.7.2. Experiment 2 In vivo viability of DNA-injected, frozen blastocysts were examined following
transfer to recipients. In this study, a total of 457 embryos was loaded into 217 straws at a maximum of five embryos per straw and frozen. The blastocysts surviving after
thawing were transferred to recipients by a non-surgical method. In the frozen group, the number of single and twin transfers per recipient was 39 and 57, respectively. In the
fresh group, single embryos were individually transferred to 82 recipients and transfers of two embryos per recipient were performed to 80 recipients. Following transfer, the
recipients were observed for signs of estrus, and pregnancy was confirmed by palpation per rectum at approximately 60 days of gestation. Transfers of DNA-injected embryos
Ž .
were carried out over 21 2 years March 1994 to September 1996 . 2.8. Identification of transgene
Chromosomal DNAs from ear and blood samples of calves were isolated as described Ž
. Ž
by Hogan et al. 1986 . Transgene was identified by a Southern blot. Genomic DNA 10
. 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
