When touched by microtubules, the female pro-nucleus was rapidly pulled towards the male pro-nucleus.
After sperm entry, the male pro-nucleus was pushed by microtubules towards the center of the egg (Fig. 2; Video S3).
Transgenic mice were generated by pro-nucleus injection (FVB/N background).
These constructs were microinjected into the pro-nucleus of the fertilized eggs from CD-1 as described (Nagy et al., 2003) Genotyping of CAG-Dkk1 transgenic mice obtained using the PCR primers CAG-pro (5′-TAGAGCCTCTGCTAACCATGTTCATGCCTT-3′) and CAG-Dkk1 (5′-TCAGCGCAAGGGTAGGGCTGGTAGTTGTCA-3′), yielding 443 bp.
The parents' genetic material is then injected into a healthy donor egg which has had its pro-nuclei removed but still has healthy mitochondria.
Put briefly, conventional IVF is performed and then, after fertilisation, the pro-nuclei of the father and mother - you can see them clearly as the two round balls inside the glass tube in the picture above - are sucked out of the developing embryo.
After being linearized with the enzymes as indicated, the transgene cassettes were respectively injected into pro-nuclei of B6/CBA F1 zygotes as described elsewhere [67].
Transgenic mice were generated by microinjection of the expression unit (NotI fragment) of the plasmid pMGD72 into pro-nuclei of C57BL/J6 x CBA F1 fertilized eggs.
The SPC-p53(273H) transgenic mice were generated by microinjection of a 6.7-kb fragment of the SPC-p53(273H) construct into the pro-nuclei of FVB/N mouse zygotes as previously reported [23].
The final construct was then linearized using XhoI, injected into the pro-nuclei of fertilized C57Bl6/C3H hybrid eggs and implanted into pseudopregnant female mice.
Microinjection of the transgene into pro-nuclei of fertilized eggs gave rise to total 30 live pups, 6 of them carried SPC/Δ3B4DG transgene with 4 of them achieved germ-line transmission.
