Nuclear transplantation in the mammalian embryo by microsurgery and cell fusion

Nuclear transplantation in the mouse embryo is achieved by using a method that combines microsurgical removal of the zygote pronuclei with the introduction of a donor nucleus by a virus-mediated cell fusion technique.

BACKGROUND OF THE INVENTION 
In recent years there has been considerable interest in determining the 
contributions to embryonic and adult phenotype by the pronuclei and 
cytoplasm of mammalian embryos. For example, hairpin-tail (T.sup.hp) in 
mice is an allele of branchyury (T) situated on chromosome 17 (linkage 
group IX) of the mouse (Johnson, D. R., Genetics 76, 795-805 (1974)). It 
appears to differ from all previously described mammalian genes in that 
the phenotype and viability of the heterozygote depend upon the parent 
from which the T.sup.hp gene is inherited. Heterozygotes with a T.sup.hp 
/+ father are short tailed and generally viable. On the other hand, the 
majority of the heterozygotes whose T.sup.hp was derived from the egg die 
in the latter half of embryogenesis (gestational days 15-16) with a few 
exceptional T.sup.hp /+ embryos surviving until birth (Johnson, D. R. 
Genet. Res. 24, 207-213 (1975); Alton A. Doctoral Dissertation, Cornell 
U., N.Y. (1982)). Those T.sup.hp /+ progeny born, however, are cyanotic 
and die within 24 hours of parturition (Johnson, D. R. (1975), supra.). 
The T.sup.hp maternal lethal effect transmitted through the ovium could be 
inherited via the cytoplasm (oogenic defect) or via the female pronucleous 
(embryogenic defect). Thus, a method for determing whether this defect is 
transmitted by the pronucleus or cytomplasm of the egg would be most 
useful. 
Another problem is that of breeding barriers. There are several different 
species of mice. Some can interbreed, while others cannot. The breeding 
barrier is caused by the plasma membrane and cytoplasm of the embryo. 
Accordingly, it would be desirable to provide a method whereby the 
breeding barrier could be overcome. 
Another area of concern is the potential extinction of certain rare 
mammalian species, an example of which is the Siberian tiger. There are 
very few of this species remaining and their reproductive performance is 
poor. Accordingly it would be desirable if a method could be devised 
whereby such species could be preserved. 
It would also be desirable to develop certain mammalian strains having 
desirable genetic characteristics provided by the pronucleus of one 
genotype and those provided by the cytoplasm of another genotype. 
Nuclear trannsplantation methods might provide a viable solution to the 
foregoing problems. Nuclear transplantation studies in the amphibian 
embryo have provided valuable information about the possible restriction 
of nuclear potential during development (Danielli, J. R., et al, Int. Rev. 
Cytol. Suppl. 9 (1979)). Similar experiments in the mammalian embryo have 
been hindered by the small size of the embryo and its sensitivity to 
microsurgical manpulation. Although some success in nuclear 
transplantation in the mouse embryo by microsurgery has been reported 
(Illmensee K. et al, Cell 23 9-18 (1981)), it is believed to be due to the 
fact that the enucleation of the recipient cell in a small number of cases 
was incomplete enabling growth. 
SUMMARY OF THE INVENTION 
This invention relates to a novel microsurgical method for nuclear 
transplantation in mammilian embryos which, in its most fundamental 
aspects comprises: 
(a) penetrating the zona pellucida of a one-cell stage mammalian donor 
embryo with an enucleation pipette without penetrating the plasma membrane 
of the donor embryo, the pipette having an internal diameter sufficiently 
large to receive the pronuclei of the donor embryo without rupturing same; 
(b) aspirating a small portion of the plasma membrane and cytoplasm and 
pronuclei of the door embryo into the pipette with the plasma membrane 
surrounding the pronuclei; 
(c) withdrawing the pipette from the donor embryo to cause the plasma 
membrane extending between the pipette and donor embryo to form a fine 
thread and the orifice made by the pipette in the zona pellucida to close 
and pinch off the plasma membrane at the site of the orifice; 
(d) introducing the pipette containing the membrane-bound pronuclei to a 
liquid suspension of inactivated Sendai virus and aspirating into the 
pipette a small quantity of the virus suspension; 
(e) penetrating the zona pellucida of a recipient mammalian embryo which 
has been enucleated by the procedure of steps (a), (b) and (c) at the site 
of enucleation without penetrating the plasma membrane, and injecting the 
virus suspension and membrane-bound pronuclei into the perivitelline space 
of the recipient embryo; 
(f) withdrawing the pipette from the recipient embryo, and 
(g) incubating the recipient embryo at about 37.degree. C. for a period of 
time to effect fusion of the pronuclei with the recipient embryo. 
The recipient embryo containing membrane-bound nuclei is incubated for a 
period of time to permit the embryo to develop to the blastocyst stage, 
and then transferred to the uterus of a pseudopregnant mammal. Prior to 
being enucleated, the embryos should be placed in Hepes-buffered Whitten 
medium containing cytochalasin B and Colcemid. 
The above-described microsurgical procedure advantageously provides an 
elegant tool for investigating solutions for the above discussed problems. 
More particularly, the method has been used successfully to investigate 
the nuclear/cytoplasmic origin of maternal T.sup.hp lethality in the mouse 
by using it in performing reciprocal nuclear transplantation between one 
cell stage embryos from T.sup.hp /+ and +/+ females. The results obtained 
show that the introduction of T.sup.hp /+ pronuclei into +/+ cytoplasm 
does not reverse the maternally inherited lethal effect of T.sup.hp and 
leads to the conclusion that in the fertilized egg, the defect responsible 
for the T.sup.hp /+ maternal lethal effect lies in the pronuclei and not 
in the egg cytoplasm. 
The microsurgical technique of the present invention may also be adapted to 
overcoming the breeding barrier problem. For instance, the embryo for one 
species could be enucleated and the pronuclei from another species could 
be introduced to the enucleated embryo, followed by incubation and cell 
fusion. After development to the blastocyst stage, the embryo may then be 
transferred to the uterus of a pseudo-pregnant female of the species which 
produced the recipient embryo. 
A procedure similar to that indicated for overcoming the breeding barrier 
problem may be used in preserving a rare mammalian species. Thus, the 
pronculei from the embryo of a Siberian tiger could be introduced into the 
enucleated embryo of a common lion species. After incubation the embryo 
could then be transferred to the uterus of a psudo-pregnant female of the 
same species which produced the recipient embryo. 
The present invention also makes possible the development of certain 
mammalian strains having the desirable genetic characteristics provided by 
the pronucleus of one genotype and those provided by the cytoplasm of 
another genotype.

DETAILED DESCRIPTION OF THE INVENTION 
Although the detailed description of the invention is with respect to 
nuclear transplantation and cell fusion in the mouse embryo, it will be 
readily apparent to those skilled in the art that the microsurgical 
procedure described is applicable to other mammalian species. 
The holding and enucleation pipettes for use in the novel microsurgical 
process can be fashioned from Pyrex capillary tubing of suitable size 
which can, if necessary be drawn to a size related to the size of the 
embryos and pronuclei. In the case of mouse embryos the tubing from which 
the enucleation pipette is fashioned can have an outer diameter of about 
1.0 mm and an inner diameter of about 0.65 mm. Using a standard pipette 
puller, e.g. a DKI vertical puller, the outer diameter can draw down to 
about 15 to 20 .mu.m. The inside diameter of the pipette after drawing 
should be sufficient to enable aspiration of the pronuclei and surrounding 
plasma membrane into the pipette without injuring the pronculei or 
rupturing the plasma membrane. The tip of the enucleation pipette should 
be beveled and the outermost portion of the bevel should be provided with 
a longitudinally extending tip to enable piercing of the zona pellucida 
(see the several figures). The bevel can be obtained on a grinding wheel, 
followed by treatment of the bevel with a solution of hydrofluoric acid 
(25 percent) and sharpening on a microforge. 
The holding pipettes may be fashioned from the same Pyrex capillary tubing 
as the enucleation pipette. The holding pipette usually can be hand-drawn 
over a burner to the desired size, e.g. 75 to 100 .mu.m for securing a 
mouse embryo. The end which comes in contact with the embryo should be 
rounded and polished (see FIGS. 2 and 4). 
Before use, the pipettes should be cleaned and sterilized. 
One cell stage embryos are obtained from oviducts excised from 
spontaneoulsy mated females on the day of vaginal plug (usually day 1). 
Cumulus cells can be dispersed in Whitten medium (Whitten, W. K., Adv. 
Biosci. 6, 129 (1971)) containing bovine hyaluronidase (500 non-filtered 
units per ml.). Before microsurgery, the embryo, usually a group of six to 
eight embryos, is incubated for 10 to 45 minutes at 37.degree. C. in an 
atmosphere of 5 percent O.sub.2, 5 percent CO.sub.2 and 90 percent N.sub.2 
in bicarbonate-buffered Whitten medium containing cytochalasin B (5 
.mu.g/ml) and Colcemid (0.1 .mu.g/ml) (McGrath, J. and Solter, D., Science 
220 1300 (1983). The cytochalasin B destroys reversibly the microfilaments 
in the embryo and the Colcemid destroys the microtubules. As a result of 
such treatment the embryo is quite flexible. Each embryo is then placed 
singly in a hanging drop of Hepes-buffered Whitten medium containing 
cytochalasin B (5 .mu.g/ml) and Colcemid (0.1 .mu.g/ml) in a Leitz or 
other suitable microscope oil chamber fitted with a micromanipulators and 
a fixed stage microscope such as a Leitz Laborlux II. All microsurgery is 
performed as described in greater detail hereinbelow. 
Sendai virus used in the microsurgical procedure of the invention may be 
obtained from the infected allantoic fluid of embryonated chicken eggs and 
inactivated with .beta.-propiolactone at a concentration of 2000 to 3000 
hemaglutinating units per millimeter (Giles, R. E. et al, In Vitro 9 103 
(1973); Neff, J. M. et al, Prac. Soc. Exp. Biol. Med 127 206 (1968); 
Graham, C. F., Acta Endiocrinol. Suppl. 153, 154 (1971). The Sendai virus 
is a flu-type virus which has been inactivated, but has intact proteins, 
and causes cell fusion. 
Following the microsurgery, the recipient embryo is washed one or more 
times and cultured to the blastocyst stage in 50 .mu.l drops of modified 
Whitten medium (Abramczuk, J., et al., Dev. Biol. 61, 378 (1977)) under 
silicone oil at 37.degree. C. in an atmosphere of 5 percent O.sub.2, 5 
percent CO.sub.2 and 90 percent N.sub.2 (Nature (London) 283) 479 (1980)). 
The embryo at the blastocyst stage may be transferred to the uterus of a 
pseudopregnant female. Also control embryos isolated at the one-cell stage 
are similarly cultured and transferred to the uteri of pseudopregnant 
females. However, the control embryos are not exposed to cytoskeletal 
inhibitors or inactivated Sendai virus. 
Turning now to the microsurgical procedure of the invention, the mouse 
embryos are incubated before and during microsurgery in cytochalasin B 
(Ilmensee, K. et al, Cell 23, 9 (1981); Hoppe, P. C., et al, Proc. Natl. 
Acad. Sci., U.S.A. 79, 1012 (1982) and 74, 5657 (1977); Modlinski, J. A., 
J. Embroyl. Exp. Morphol. 60 153 (1980)) and Colcemid. Each embryo is then 
secured by a holding pipette and the zona pellucida is penetrated with an 
unucleation pipette of the type heretofore described. Penetration of the 
plasma membrane, however, is avoided and the pipette is advanced to a 
position adjacent each pronucleus. Upon aspiration, a small portion of the 
plasma membrane and surrounding cytoplasum are drawn into the pipette, 
followed by the pronucleus. The pipette containing the entire pronucleus 
is then moved to a point adjacent the second pronucleus and the latter is 
similarly aspirated. 
Referring to FIG. 1, as the enucleation pipette is withdrawn, the plasma 
membrane which extends into the pipette and surrounds the pronuclei 
stretches into a fine thread or cytoplasmic bridge which is then pinched 
off by the zona pellucida through closing of the orifice made by the 
pipette therein (see arrow FIG. 1 and FIG. 2). 
The pipette, which contains the membrane-bound pronuclei (pronuclear 
karyoplast), is moved in contact with the Sendai virus suspension, 
described previously, and a small volume of the virus suspension, 
generally on the order of the volume of the pronuclear kyroplast, is 
aspirated into the pipette. 
The pipette containing both the pronuclear kyroplast and Sendai virus is 
moved to a third drop containing an embryo enucleated previously using the 
above-described procedure. The zona pellucida of the latter embryo is 
penetrated at the previous site of enucleation, and the virus suspension 
and pronuclear karyoplast are injected into the perivitelline space (see 
FIG. 3). After withdrawal of the pipette, the embryo is incubated at 
37.degree. C. Fusion of the pronuclear karyoplast with the enucleated 
embryo generally occurs during the first hour of incubation (see FIG. 4). 
The embryo is then cultured in vitro for approximately 5 days during which 
the embryo develops into the blastocyst stage. The embryo is then 
transferred to the uterus of a pseudopregnant female where gestation takes 
place resulting in the birth of a progeny. Alternatively the manipulated 
embryo is transferred to the oviduct of a pseudopregnant female 
immediately after the nuclear tranfer (McGrath, J. and Solter, D., Nature 
(London) 308 550 (1984). 
A study was made to determine the efficiency of the nuclear transplant 
procedure described above. The study involved a total of 73 embryos 
divided among three different mouse genotypes. The results of the study 
are set forth in Table 1, below: 
TABLE 1 
______________________________________ 
Efficiency of the Nuclear Transplatation Technique 
Karyoplast 
Genotype Enucleation.sup.1 
Injection.sup.2 
Fusion.sup.3 
______________________________________ 
C3H/HeJ 24 of 26 24 of 24 23 of 24 
C57BL/6J 35 of 35 34 of 35 34 of 34 
ICR 11 of 12 10 of 11 10 of 10 
Total (%) 
70 of 73 (96) 
68 of 70 (97) 
67 of 68 (99) 
______________________________________ 
.sup.1 Number of embryos surviving microsurgical removal of both male and 
female pronuclei per total number of embryos. 
.sup.2 Number of pronuclear karyoplasts surviving injection into the 
perivitelline space of the recipient embryo per total number of 
karyoplasts injected. 
.sup.3 Number of pronuclear karyoplasts fusing with the recipient embryo 
per total number of karyoplastinjected embryos. 
Referring to Table 1, it can be seen that of 73 experimental embroys, 70 
(96%) were successfully enucleated, and of the 70 pronuclear karyoplasts 
obtained, 68 (97%) were successfully introduced (with Sendai virus) into 
the perivitelline space of enucleated zygotes. After incubation at 
37.degree. C., 67 (99%) of these karyoplasts fused to the plasma membrane 
of the ovium. The overall efficiency of the transplantation method of the 
invention was, therfore, 91 percent. 
After microsurgery, experimental and control embryos were cultured for 5 
days and the number of embryos successfully developing to the bastocyst 
stage was determined. Of 34 control embryos, all developed to the morula 
or bastocyst stage (Table 2). Similarly, of the 67 experimental embryos, 
64 (96%) developed to the morula or blastocyst stage. Transfer of the 34 
control embryos to the uteri of pseudopregnant females resulted in the 
birth of five progeny (15%), three of which survived to adulthood. 
Transfer of the 64 experimental embryos to the uteri of pseudopregnant 
females resulted in the birth of ten progeny (16%), seven survived to 
adulthood. These seven offspring all displayed the coat color phenotype of 
the donor nuclei, and five were fertile. 
Thus the technical manipulations involved in transferring pronuclei from 
one zygote to another did not significantly affect the ability of embryos 
to undergo normal development. The high frequency of developmental arrest 
in both experimental and control groups after the implantation procedure 
may have resulted from the in vitro culture period before implantation, 
since the intrauterine transfers of 22 carrier blastocytsts that had 
developed in vivo resulted in the birth of 17 progeny. Reciprocal 
pronuclear transplantations between genetically distinct one-celled 
embryos may be used to define the degree to which maternally inherited 
cytoplasmic components persist. 
TABLE 2 
______________________________________ 
Development of Control and Nuclear-Transplant Embryos. 
The Subscripts n and c Refer to the Strain Origin 
of the Nucleus and the Cytoplasm, respectively. 
Developmental stage by day 5 
in vitro (number of embryos) 
Blasto- 
Number 
Group and Strain 
Arrested Morula cyst Born.sup.4 
______________________________________ 
Control 
C3H/HeJ 0 3 11 0 
C57BL/6J 0 1 13 4 
ICR 0 1 5 1 
Total 0 5 29 5 
Nuclear-transplant 
C3H/He.sub.n to C57B1/6J.sub.c 
0 0 23 4 
C57BL/6J.sub.n to C3H/He.sub.c 
0 1 23 3 
ICR.sub.n to C57BL/6J.sub.c 
1 0 9 2 
C57B/6J.sub.n to ICR.sub.c 
2 0 8 1 
Total 3 1 63 10 
______________________________________ 
.sup.4 Number of offspring born after transfer of morulae and blastocysts 
into the uteri of females of the third day of pseudopregnancy. 
The microsurgical procedure of this invention was also used in a study to 
determine whether T.sup.hp lethality in the mouse is a nuclear of a 
cytoplasmic defect. 
Using the microsurgical procedure of this invention, one cell-stage embryos 
obtained from +/+ albino females mated to +/+ albino males were used in 
reciprocal nuclear transplantations with one cell-stage embryos obtained 
from pigmented T.sup.hp /+ females previously mated to pigmented +/+ 
males. Nuclear transplant embryos were transferred to the oviducts of day 
1 pseudopregnant females and allowed to develop to term. The number, coat 
color phenotype and tail length of the progeny were observed as T.sup.hp 
causes a shortening of the tail when heterozygous. 
T.sup.hp /+ embryos were obtained from brown or agouti T.sup.hp /+ females 
mated to C57BL/6J males, while +/+ embryos were obtained from matings of 
outbred albino CD-1 (Charles River) males and females. Pseudopregnant 
females were obtained from matings of CD-1 females with vasectomized and 
proven sterile CD-1 males. All matings were spontaneous. Female mice were 
sacrificed on the day of vaginal plug detection (day 1 of pregency) and 
their oviducts excised. The ampulla of the oviduct was punctured with a 
watchmaker forceps and the released embryos were freed from surrounding 
cumulus cells by incubation in modified Whitten's medium (Whitten, W. K. 
supra.) containing 500 u/ml bovine hyaluronidase (Sigma). Nuclear 
transplatation was performed as previously described. Among 545 embryos, 
535 (96%) were successfully enucleated and of the 524 pronuclear 
karyoplasts, 494 (94%) were successfully injected along with inactivated 
Senda virus into the perivitelline space of enucleated recipient embryos. 
403 (.about.80%) of the pronuclear karyoplast: enucleated cytoplasm pairs 
underwent fusion. The latter were transferred on the day of microsurgery 
to the oviducts of day 1 pseudopregnant females using a small bore glass 
pipette. The results of these experiments are set forth in Table 3. 
TABLE 3 
______________________________________ 
Phenotype of Nuclear Transplant Offspring 
No. of 
successful 
No. of progeny 
Nuclear Cytoplasmic 
nuclear Normal Short 
genotype genotype transplants 
Tail Tail 
______________________________________ 
T.sup.hp /+, +/+ 
+/+ 206 16 2 
+/+ T.sup.hp /+, +/+ 
197 45 0 
______________________________________ 
Referring to Table 3, of 197 successful nuclear transplant embryos in which 
wild-type pronuceli from albino females mated to albino males were 
introduced into the cytoplasm of enucleated embryos from pigmented 
T.sup.hp /+ females, 45 (20 females and 25 males) normal-tailed albino 
progeny resulted (23%). Among 206 successful nuclear transplant embryos in 
which pronuclei from pigmented T.sup.hp /+ females mated to pigmented 
males were introduced into the cytoplasm of enucleated embryos from albino 
females, 18 (10 females and 8 males) pigmented progeny were born (9%). 
Thus, a significantly greater proportion of nuclear transplant embryos, in 
which the egg cytoplasm was derived primarily from T.sup.hp /+ females, 
developed to term (23%) than those embryos in which egg cytoplasm was 
derived primarily from +/+ females (9%) (X.sup.2 =8.99; P&lt;0.01). These 
data show no evidence that ovum cytoplasm from T.sup.hp /+ females 
inhibits normal embryogenesis. 
Since the T.sup.hp /+ females were mated to +/+ males, half of the nuclear 
transplant embryos should be T.sup.hp /+ (short-tailed) and half +/+ 
(normal-tailed). Among the 18 progeny which resulted from the introduction 
of pronuclei from T.sup.hp /+ females into +/+ cytoplasm (Table 3), a 
significantly greater number of normal-tailed progeny were born (16) than 
short-tailed progeny (2) (X.sup.2 =9.38; P&lt;0.01). Of the 16 normal-tailed 
progeny, 2 were dead at birth while the remaining 14 were viable and 
survived to adulthood. Of the two T.sup.hp /+ progeny, one was dead at 
birth while the other was edematous, cyanotic and died within 24 hours of 
parturition. The number of viable normal-tailed progeny (14) is 
significantly greater than the number of viable short-tailed progeny (0) 
(X.sup.2 =12.06; P&lt;0.001). Because the introduction of T.sup.hp /+ 
pronuclei into wild-type cytoplasm did not result in the birth of viable 
T.sup.hp /+ progeny it can be concluded that T.sup.hp -lethality is 
inherited as a nuclear, not cytoplasmic, defect. 
The several technical papers identified in this specification are 
incorporated by reference herein in their entirety.