2. Materials and methods
2.1. Mice and egg collection A total of 130 B6C3F female mice of 7 weeks of age were used for the collection of
1
fertilized eggs, and twelve 10–12-week-old ICR female mice served as the foster Ž
mothers. Mice were purchased from a commercial breeding farm Sankyo LaboService, .
Japan and were maintained under a regulated light cycle of 14 h light and 10 h dark. The present experiments were carried out according to the guide of use and care for
laboratory animals, College of Agriculture, The University of Tokyo.
2.2. DNA constructs for microinjection Two kinds of gene constructs were used for microinjection. The 4.6 kb mWAPrhGH
Ž .
fusion gene consisted of 2.1 kb of human growth hormone hGH structural gene that flanked by 2.5 kb of the 5
X
non-coding DNA region of the mouse whey acidic protein Ž
.
A
mWAP gene, and the 8.8 kb human grb-globin fusion gene where 3.3 kb of
Ag-globin gene was linked to 5.5 kb of b-globin gene. These gene constructs have been Ž
previously used for generating transgenic mice in our laboratory Katsube et al., 1993; .
Tojo et al., 1993 , and have no EcoRI site within their sequences. 2.3. Egg collection and microinjection
The collection of pronuclear-stage eggs, culturing the embryos and DNA microinjec- tion were carried out according to the standard protocol described by Hogan et al.
Ž .
Ž 1994 . Briefly, after removing the cumulus cells of F hybrid eggs obtained by mating
2
. B6C3F = B6C3F adult , the eggs were placed in a microdrop of M16 medium which
1 1
was covered with paraffin oil, and cultured in a CO incubator until they were used for
2
the microinjecion. Microdrop of the M2 medium containing zygotes and that of the Ž
Ž .
DNA injecting buffer 10 mM Tris–HCl, pH 8.0, 1 mM EDTA or the DNA–enzyme mixture were placed separately on a culture dish and covered with mineral oil.
Pronuclear microinjection was performed under a Nikon Diaphot inverted microscope with Nomarski differential interference contrast optics. In the first experiment, to
determine the optimal amount of enzyme for the co-injection with DNA, 727 zygotes were given pronuclear microinjections of various amounts of EcoRI or of the buffer
Ž only. In the second experiment, approximately 2 pl of DNA 300–350 copies of each of
. Ž
y8
. genes solution, mixed without or with EcoRI 5 = 10
Urnucleus was injected into the male pronucleus. The restriction enzyme, EcoRI was diluted with a DNA-injecting
buffer and to make the solutions of various concentrations of RI. One part of the enzyme solution was mixed with nine parts of the solution, and the DNA construct was dissolved
at a concentration of 2 mgrml in the DNA-injecting buffer. The entire mouse genomic
Ž .
Ž .
DNA 3 pg in the nucleus Hogan et al., 1994 could be completely digested with 2 = 10
y5
U of EcoRI.
2.4. Detection of the transgene Approximately 370 zygotes microinjected with the DNA–enzyme mixture or the
buffer containing DNA alone were cultured in vitro until they developed to blastocyst stage. The presence of integrated foreign genes in the single embryos was detected
Ž .
according to the method described by Seo et al. 1997 . Embryos were washed twice in Ž
PBS and twice 1 = PCR buffer pH 8.4; 20 mM Tris–HCl, 50 mM KCl, 1.5 mM .
MgCl . They were next transferred into 0.5 ml Eppendorf tubes containing 2 ml of 1
2
Ž =
PCR buffer supplemented with 0.05 mgrml Proteinase K Sigma, St. Louis, MO, .
Ž .
Ž . Ž
USA , 1.7 mM sodium dodecyl sulfate SDS and 20 mM dithiothreitol DDT Wako,
. Osaka, Japan ; they were then incubated for 1 h at 378C, and subsequently for 5 min at
958C. Genomic DNA extracted from a single embryo was digested with 200 mU DpnI Ž
. Gibco BRL, NY, USA at 378C for 1 h in a final concentration of 20 mM Tris–HCl
Ž .
pH 7.4 , 5 mM MgCl , 50 mM KCl and then incubated for 10 min at 658C. A 250-ml
2
Ž .
aliquot of Bal31 Takara Shuzo, Shiga, Japan was added to the tube containing 20 mM Ž
. Tris–HCl pH.8.0 , 600 mM NaCl, 12 mM CaCl , 12 mM MgCl , 1 mM EDTA, and
2 2
then incubated sequentially for 1 h at 308C and for 10 min at 708C. DNA was recovered using the glassmilk method according to the protocol recommended by the manufacturer
Ž .
Geneclean II Kit, Bio 101; CA, USA . Briefly, a stock solution of NaI was added to the Ž
. Ž
DNA sample 3:1,vrv ; this was followed by the addition of a glassmilk suspension 0.2 .
mlrtube and the resultant mixture was incubated at 48C for 5 min. The glassmilk–DNA complex was then vortexed for about 5 s and washed 3 times with ice-cold washing
buffer. Finally, DNA was eluted into water and subjected to PCR amplification. Approximately 230 injected zygotes were transferred into the oviducts of 12 pseudo-
pregnant mice. The transgenic mice were detected by analyzing DNA extracted from the
Ž .
tail tip of 3–4-week-old pups Hogan et al., 1994 by PCR method. PCR was performed Ž
. Ž
. as follows: the reaction mixture 59 ml contained template DNA 250 ng , Tris–HCI
Ž .
Ž .
Ž .
Ž .
20 mM; pH 8.4 , KCI 50 mM , MgC1 1.5 mM deoxyribonucleotides 200 mM ,
2
. Ž
. each of ddATP, ddTT, ddCTP and ddGTP , primers 1 mM , and Taq DNA polymerase
Ž .
2.5 U; Gibco BRL . After denaturing DNA at 948C for 4 min, PCR was carried out as follows: and 35 cycles of 1 min at 948C, 1 min at 608C, and 2 min at 728C for
amplifying the specific sequences of the mWAPrhGH gene; 30 cycles of 1 min at 948C, 1 min at 608C and 1 min at 728C for the human Agrb-globin fusion gene. In the last
cycle, the extension time was prolonged to 4 min for both of the two sets of primers.
Ž .
PCR products 10 ml were applied onto 1.0 agarose gel, electrophoresed in TAE buffer, stained with ethidium bromide and photographed under ultraviolet light. The
primers used to amplify the specific sequences of transgenes were as follows: 5
X
-CTA- TTC-CGA-CACCCT-CCA-ACA-G-3
X
and 5
X
-ACA-ACG-ATG-ACG-CAC-TAC-TCA- A-3
X
, spanning exon IV and V of the hGH gene, which produced a 649 bp fragment, and 5
X
-CAA-GACAGG-TTT-AAG-GGG-GCC-A-3
X
and 5
X
-ACA-TCA-AGG-GTC-CCA- TAG-ACT-C-3
X
for the human globin gene, which produced a 361 bp fragment. 2.5. Statistical analysis
Ž
2
. Chi-square x
test was performed for statistical evaluation of the results.
3. Results
Kategori