www.elsevier.comrlocateranireprosci
Co-injection of restriction enzyme with foreign
DNA into the pronucleus for elevating production
efficiencies of transgenic animals
B.B. Seo
a,1, C.H. Kim
a, K. Yamanouchi
a, M. Takahashi
b,
T. Sawasaki
c, C. Tachi
d, H. Tojo
a,)a
Laboratory of Applied Genetics, Graduate School of Agricultural and Life Sciences, The UniÕersity of Tokyo,
Tokyo, 113-8657, Japan
b
Laboratory of Veterinary Physiology, Graduate School of Agricultural and Life Sciences, The UniÕersity of
Tokyo, Tokyo, 113-8657, Japan
c
Experimental Station for Bio-animal Science, The UniÕersity of Tokyo, Iwama-machi,
Ibaraki 319-0106, Japan
d
Laboratory of DeÕelopmental and ReproductiÕe Biotechnology, School of Veterinary Medicine and Life
Sciences, Azabu UniÕersity, Fuchinobe, Sagamihara, 229-0006, Japan
Received 21 September 1999; received in revised form 21 February 2000; accepted 20 March 2000
Abstract
The microinjection method for production of transgenic farm animals requires specialized techniques and results in intolerably low production efficiencies. We investigated whether or not co-injection of foreign DNA constructs with restriction endonuclease into the pronucleus of mouse zygotes would improve the integration frequencies of foreign DNA into the host genome. Two kinds of DNA constructs that have no EcoRI site in their sequences were used for co-microinjec-tion. With reference to the results of experiments in which EcoRI alone was injected at various
amounts varying from 10y9to 10y5Urnucleus, the amount of 5=10y8Urnucleus that showed
survival rate of 60.6% was used for the co-injection with DNA. Successful transgenesis of co-injected embryos was identified by DpnI–Bal31 digestion method for single embryos and by PCR method for pups born, respectively. The overall efficiency for the integration of foreign DNA
)Corresponding author. Tel. :q81-3584-15194; fax:q81-3584-18191.
Ž .
E-mail address: [email protected] H. Tojo .
1
Present address: Division of Biochemistry, Department of Molecular and Experimental, Medicine. The Scripps Research Institute, La Jolla, CA 92037 USA.
0378-4320r00r$ - see front matterq2000 Elsevier Science B.V. All rights reserved. Ž .
in single embryos and live-born pups obtained by the co-injection procedures were 17.9% compared with 9.1% obtained by the injection of DNA alone. The results suggest that co-injection of foreign genes with restriction enzyme may elevate the integration rate of foreign genes into host
genomes.q2000 Elsevier Science B.V. All rights reserved.
Keywords: Co-injection; Transgenic mice; Restriction enzyme; PCR; Transgenesis
1. Introduction
Microinjection of foreign DNA into the pronuclei of fertilized eggs has been extensively employed for the production of transgenic animals; however, this method for gene transfer requires an extremely specialized technique. In addition to the technical obstacle, the rate of successful incorporation of the exogenously introduced genes into the host genome has been intolerably low, particularly in farm animals. Approximately
Ž
5% of mouse eggs microinjected with foreign DNA result in transgenic mice Brinster et
. Ž
al., 1985 , while the efficiency is 0–5% in swine, and less than 1% in sheep Pursel et
. Ž .
al., 1989 and cattle Krimpenfort et al., 1991; Hill et al., 1992 .
Several attempts have been made to simplify microinjection technique and improve
Ž .
the efficiency of integration of foreign DNA. Page et al. 1995 attempted to produce transgenic mice with the cytoplasmic injection of DNA mixed with polylysine, which
Ž
has been used to transfect some mammalian cell lines Felgner et al., 1987; Behr et al., .
1989 . They reported that about 12.8% of the pups born from zygotes cytoplasmically microinjected with a polylysinerDNA mixture were transgenic, whereas no transgenic pups were born from the microinjection of DNA alone into the cytoplasm. It has generally been believed that the opportunity for foreign DNA integrated into the genome occurs only when host DNA molecules are cleaved as a result of the stimulation caused
Ž .
by the microinjection and rejoined during the repair Brinster et al., 1985 .
Ž .
Phillips and Morgan 1994 induced the DNA double-strand breaks in the
endoge-Ž .
nous gene in cultured Chinese hamster ovary CHO cells by electroporating the restriction endonuclease into the cells, and they found that blunt-end DNA double-strand
Ž .
breaks can induce illegitimate nonhomologous recombination in mammalian
chromo-Ž .
somes. Brenneman et al. 1996 demonstrated that the intrachromosomal homologous recombination in human cells was stimulated by the introduction of foreign DNA together with site-specific endonuclease. Thus, it appears that the introduction of certain restriction endonuclease into cultured mammalian cells may significantly stimulate the DNA recombination between the foreign DNA and the host genomic DNA. Although the precise details of the mechanisms of integration are still unclear, it has been proposed that the DNA integration processes in fertilized eggs and cultured cells have
Ž .
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 of1 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 5X 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. Ž1994 . Briefly, after removing the cumulus cells of F hybrid eggs obtained by mating. 2 Ž
.
B6C3F1=B6C3F adult , the eggs were placed in a microdrop of M16 medium which1
was covered with paraffin oil, and cultured in a CO incubator until they were used for2
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 .
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 22 ml of 1 Ž
=PCR buffer supplemented with 0.05 mgrml Proteinase K Sigma, St. Louis, MO,
. Ž . Ž . Ž
USA , 1.7mM 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 ŽGibco BRL, NY, USA at 37. 8C for 1 h in a final concentration of 20 mM Tris–HCl ŽpH 7.4 , 5 mM MgCl , 50 mM KCl and then incubated for 10 min at 65. 2 8C. A 250-ml
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, and2 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 ŽGeneclean 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 Ž20 mM; pH 8.4 , KCI 50 mM , MgC1. Ž . 2 Ž1.5 mM deoxyribonucleotides 200. Ž mM ,.
. Ž .
each of ddATP, ddTT, ddCTP and ddGTP , primers 1mM , and Taq DNA polymerase Ž2.5 U; Gibco BRL . After denaturing DNA at 94. 8C 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: 5X -CTA-TTC-CGA-CACCCT-CCA-ACA-G-3X and 5X -ACA-ACG-ATG-ACG-CAC-TAC-TCA-A-3X, spanning exon IV and V of the hGH gene, which produced a 649 bp fragment, and 5X-CAA-GACAGG-TTT-AAG-GGG-GCC-A-3X and 5X -ACA-TCA-AGG-GTC-CCA-TAG-ACT-C-3X for the human globin gene, which produced a 361 bp fragment.
2.5. Statistical analysis
Ž 2.
3. Results
3.1. Effect of EcoRI microinjected into the pronucleus on embryo surÕiÕal
To determine the optimal amount of the enzyme for the co-injection, we injected EcoRI alone in varying amounts ranging from 10y9 Urnucleus to 10y5rnucleus, and
examined the survival rate of the embryos. The buffer used for dissolving DNA was injected as the control. Table 1 shows the number of embryos that survived and developed in vitro afte rmicroinjection of various amounts of EcoRI into the pronucleus
Ž .
of zygotes. A sufficiently high proportion 71% of the eggs given the injection of the buffer alone developed into the morularblastocyst stage in vitro. Approximately 70% of the embryos given the injection of 10y9 Urnucleus developed into the morularblasto-cyst stage. Injection of 10y6 Urnucleus of EcoRI resulted in a remarkable reduction in the viability of the injected eggs, though 12% of the injected embryos developed to the
Ž . y5
four-cell stage data not shown . The concentrations more than 10 Urnucleus did not result in any viable embryos. Injections of amounts of 5=10y8 and 10y7 Urnucleus,
resulted in survival rates of 60.6% and 48.5% of the embryos, respectively. There was a good linear correlation between the amount of the enzyme injected and the mean survival rate of the injected eggs. We used 5=10y8Urnucleus in the subsequent series
of experiments in which the enzyme was co-injected with DNA because this
concentra-Ž .
tion gave reasonable survival rate 60.6% before drastic reduction.
3.2. Effects of the co-injection of DNA constructs with the restriction endonuclease on the integration rate of foreign genes
The results of co-injecting DNA constructs with restriction endonuclease are shown in Table 2. In this series, each of the two gene constructs was dissolved in the injection
Table 1
In vitro development of mouse zygotes that were given pronuclear injections of various amounts of EcoRI
Ž .
Treatment No. of No. of No. of developed embryos %
a
trials eggs used Morulae Blastocyst Total
Ž . Ž . Ž .
Non-injection 9 294 7 2.3 275 93.5 282 95.9
b Ž . Ž . Ž .
Injection of buffer 2 107 15 14.0 61 57.0 76 71.0
y9 Ž . Ž . Ž .
Amounts of injected 10 2 63 8 12.7 36 57.1 44 69.8
y8
The embryos did not develop beyond morula.
b Ž .
10 mM Tris–HCl pH 8.0 , 1 mM EDTA.
c Ž .
()
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Table 2
Effects of the co-injection of foreign genes with or without EcoRI into the pronucleus of mouse zygotes on transgenesis in single embryos and pups born Each value of gene injections with or without the enzyme includes the results of two or three trials.
Injected foreign genes Gene with EcoRI Gene alone Overall efficiency
mWAPrhGH Human Agrb-globin mWAPrhGH Human Agrb-globin Gene with EcoRI Gene alone
a
Ž . Ž . Ž . Ž . Ž . Ž . Ž .
No. of transgenic embryos % 6r36 16.7 4r21 19.0 2r21 9.5 3r23 13.0 10r57 17.5 5r44 11.4
b
Ž . Ž . Ž . Ž . Ž . Ž . Ž .
No. of transgenic pups % 0r5 0 2r5 40.0 0r6 0 0r5 0 2r10 20.0 0r11 0
c c
Ž . Ž . Ž . Ž . Ž . Ž . Ž .
Overall of transgenesis % 6r41 14.6 6r26 23.1 2r27 7.4 3r28 10.7 12r67 17.9 5r55 9.1
a Ž .
DNA extracted from the single late–stage embryo was digested with DpnI and Bal31, and was analyzed by PCR Soe et al., 1997 .
b
DNA extracted from tail tip of pups born was analyzed by PCR.
Ž . Ž y8 .
buffer 1mgrml containing EcoRI equivalent to 5=10 Urnucleus and injected into a zygote pronucleus. The human Agrb-globin fusion gene has the ClalrSal l restriction ends, but does not have EcoRI site within its construct. The embryos that survived the injection were cultured in vitro until they developed into the morular blastocyst stage. Fig. 1 shows representative photographs of the agarose-gel elec-trophoresis of PCR products of DNA extracted from transgenic single embryos follow-ing the co-injection of DNA with EcoRI. The embryos that integrated the foreign gene
Ž .
were identified by the Dpnl–Bal31 digestion method described by Seo et al. 1997 , while the transgenic pups were identified by the standard PCR method. Co-injection of
Fig. 1. Representative agarose–gel electrophoretograms of PCR products using the DNA extracted from the single blastocysts as the templates after a combined digestion with DpnI endonuclease and Bal31 exonuclease
Žsee Materials and Methods . Arrows indicate the positions of PCR products amplified by the specific primers..
mWAPrhGH and humanAgrb-globin indicate the gene constructs that injected to the pronucleus of zygotes, alone or with EcoRI. M, molecular weight markers. The same PCR products were detected by analysis of
Ž .
the genes with EcoRI appeared to have resulted in the elevation of the rate of integration in the embryos examined: the overall efficiency of the embryos that integrated the foreign genes was 17.5% for the co-injection group and 11.4% for the DNA alone group. Seven of 12 recipients with the transfer of the injected embryos gave birth to 21 pups. The overall efficiency of transgenesis in pups with the co-injection was 20.0%, while no transgenic pups were born when DNA was injected alone. The overall efficiency of the integration of foreign DNA in the embryos and live-born pups obtained by the present co-injection procedures was 17.9% compared with 9.1% obtained by the injection of DNA alone. When the DNA injection with or without EcoRI was compared regarding overall efficiency of transgenesis, the co-injection group was better than the control group, although the difference between the two groups, was not statistically
Ž Ž 2 ..
significant 0.1-P-0.2 x test .
4. Discussion
Ž .
Although Brinster et al. 1985 proposed a working hypothesis that spontaneous breaks occur in chromosomes, possibly accelerated by the microinjection procedure, and that these breaks function as the sites of the integration of linear DNA by ligation reactions, little is known about the true mechanisms of integration of foreign DNA into the genome. In the mammalian genome, double-strand breaks of DNA can occur during
Ž .
cellular proliferation or as a result of exposure to DNA-damaging agents Ward, 1988 . Understanding the mechanisms underlying the repair of double-strand breaks and how these processes are carried out at, both the DNA and chromosomal levels will help to clarify the mechanisms of transgene integration into host genomes. Recently, the HO or I-Sce-I endonuclease of yeast has been shown to be expressed in mammalian cells and to stimulate homologous recombination between a transfected DNA and a chromosomal
Ž .
locus Phillips and Morgan, 1994; Brenneman et al., 1996 .
Ž .
Godwin et al. 1994 electrophoresed a restriction enzyme, PaeR71, into hamster cells containing heteroallelic reporter genes, but they detected only nonhomologous repair of the chromosomal cleavage sites and no homologous recombination between
Ž .
chromosomes. Brenneman et al. 1996 reported interesting results of their experiments showing that the treatment of human cell lines with the restriction endonuclease XbaI, which has a recognition site within the treated region of HPRT homology, increased the frequency of homologous recombination by more than tenfold.
We have attempted to investigate whether co-injection of foreign DNA constructs with restriction endonuclease into the pronucleus of mouse zygotes improve the integra-tion frequencies of foreign DNA into host genomes. The amounts of DNA with a haploid number of chromosome present in the pronucleus of a fertilized ovum is
Ž .
approximately 6 pg Hogan et al., 1994 . It can be suggested that the haploid DNA Ž6 pg would be completely digested by treatment of the pronucleus with 6. =10y6
necessary for the complete digestion of haploid mouse DNA, was injected into the pronucleus, none of the injected embryos could develop beyond the four-cell stage in vitro. We postulated that the host DNA was almost completely digested by the enzyme, beyond repair. We chose, therefore, 5=10y8 Urnucleus, which gave reasonable
Ž .
survival rate 60.6% , for the co-injection amount.
In our laboratory, we have produced a number of transgenic mice, in which a variety Ž
of DNA constructs had been introduced Katsube et al., 1993; Tojo et al., 1993; Ikeda et . al., 1994; Hase et al., 1996; Kano et al., 1998; Inuzuka et al., 1999; Kato et al., 1999 . The mean efficiency of transgenesis in the pups born was less than 11%. Therefore, the present results suggest that co-injection of foreign genes with restriction enzyme possibly elevates the integration rate of foreign genes into host genomes, though the difference between the two injections with and without the restriction enzyme was not
Ž .
statistically significant 0.1-P-0.2 and additional experimentation is needed in the future.
Acknowledgements
Ž This work was supported in part by a Grant-in-Aid for Scientific Research No.
.
07556083 from The Ministry of Education, Science, Sports, and Culture of Japan.
References
Behr, J.P., Demmeneix, B., Leoffler, J.P., Perez-Mutul, J., 1989. Efficient gene transfer into mammalian primary endocrine cells with lippolyamine-coated DNA. Proc. Natl. Acad. Sci. U. S. A. 86, 6982–6986. Bishop, J.O., Smith, P., 1989. Mechanism of chromosomal integration of microinjected DNA. Mol. Biol. Med.
6, 283–298.
Brenneman, M., Gimble, F.S., Wilson, J.H., 1996. Stimulation for intrachoromosomal homologous recombina-tion in human cells by electroporarecombina-tion with site-specific endonuclease. Proc. Natl. Acad. Sci. U. S. A. 85, 836–840.
Brinster, R.L., Chen, H.Y., Traumbauer, M.E., Yagle, M.K., Palmiter, R.D., 1985. Factors affecting the efficiency of introducing foreign DNA into mice by microinjection. Proc. Natl. Acad. Sci. U. S. A. 82, 4438–4442.
Felgner, P.L., Gadek, T.R., Holm, M., Roman, R., Chan, H.W., Wenz, M., Northrop, J.P., Ringold, G.M., Danielsen, M., 1987. Lipofectin: a highly efficient, lipid-mediated DNA-transfection procedure. Proc. Natl. Acad. Sci. U. S. A. 84, 7413–7417.
Godwin, A.R., Bollag, R.J., Christie, D.M., Liskay, R.M., 1994. Spontaneous and restriction enzyme-induced chromosomal recombination in mammalian cells. Proc. Natl. Acad. Sci. U. S. A. 91, 12554–12558. Hase, W., Seo, B.B., Tojo, H., Tanaka, S., Tachi, C., 1996. Transgenic mice over expressing foreign whey
Ž .
acidic protein WAP genes,driven by metallothionein promoter. J. Reprod. Dev. 42, 265–271.
Hill, K.G., Curry, J., DeMayo, F.J., Jones-Diller, K., Slapak, J.R., Bondioli, K.R., 1992. Production of
Ž .
transgenic cattle by pronuclear injection. Theriogenology 37, 222, Abdtr. .
Hogan, B., Beddington, R., Costantini, F., Lacy, E., 1994. Manipulating the Mouse Embryo. A Laboratory Manual. 2nd edn. Cold Spring Harb., Cold Spring Harb. Lab. Press, NY.
Inuzuka, H., Yamanouchi, K., Tachi, C., Tojo, H., 1999. Expression of milk protein genes is induced directly by exogenous STAT5 without prolactin-mediated signal transduction in transgenic mice. Mol. Reprod. Dev. 54, 121–125.
Kano, K., Tojo, H., Yamanouchi, K., Soeta, C., Tanaka, S., Ishii, S., Tachi, C., 1998. Skeletal muscle of transgenic mice expressing human snoN, a homologue of c-ski. J. Reprod. Dev. 44, 253–260.
Kato, K., Yamanouchi, K., Ikawa, M., Okabe, M., Nito, M., Tojo, H., 1999. Efficient selection of transgenic mouse embryos using EGFP as a marker gene. Mol. Reprod. Dev. 54, 43–48.
Katsube, K., Tojo, H., Sakaguchi, Y., Fukumaki, Y., 1993. Human fetal-adult globin switching in transgenic mice; persistent expression of the Gg-globin gene in the Japanese HPFH. Biochem. Biophys. Res. Commun. 194, 97–107.
Krimpenfort, P., Rademakers, A., Eyestone, W., van der Schans, A., van den Broek, S., Kooiman, P., Kootwijk, E., Platenburg, G., 1991. Generation of transgenic dairy cattle using in vitro embryo production. Biotechnology 9, 844–847.
Page, R.L., Bulter, S.P., Subramanian, A., Gwazdauskas, F.C., Johnson, J.L., Velander, W.H., 1995. Transgenic mice by cytoplasmic injection of polylysinerDNA mixtures. Transgenic Res. 4, 353–360. Phillips, J.W., Morgan, W.F., 1994. Illegitimate recombination induced by DNA double-strand breaks in a
mammalian chromosome. Mol. Cell. Biol. 14, 5794–5803.
Pursel, V.G., Pinkert, C.A., Miller, K.F., Bolt, D.J., Campbell, R.G., Palmiter, R.D., Brinster, R.L., Hanllner, R.E., 1989. Genetic engineering of livestock. Science 244, 1281–1288.
Seo, B.B., Kim, C.H., Tojo, H., Tanaka, S., Yamanouchi, K., Takahashi, M., Sawasaki, T., Tachi, C., 1997. Reprod. Fert. Dev. 9, 263–269.
Tojo, H., Tanaka, S., Matsuzawa, A., Takahashi, M., Tachi, C., 1993. Production and characterization of transgenic mice expressing a hGH fusion gene driven by the promoter of mouse whey acidic protein
ŽmWAP putatively specific to mammary gland. J. Reprod. Dev. 39, 145–155..
