Generation of cloned mice and nuclear transfer embryonic stem cell lines from urine-derived cells

Abstract

Cloning animals by nuclear transfer provides the opportunity to preserve endangered mammalian species. However, there are risks associated with the collection of donor cells from the body such as accidental injury to or death of the animal. Here, we report the production of cloned mice from urine-derived cells collected noninvasively. Most of the urine-derived cells survived and were available as donors for nuclear transfer without any pretreatment. After nuclear transfer, 38-77% of the reconstructed embryos developed to the morula/blastocyst, in which the cell numbers in the inner cell mass and trophectoderm were similar to those of controls. Male and female cloned mice were delivered from cloned embryos transferred to recipient females, and these cloned animals grew to adulthood and delivered pups naturally when mated with each other. The results suggest that these cloned mice had normal fertility. In additional experiments, 26 nuclear transfer embryonic stem cell lines were established from 108 cloned blastocysts derived from four mouse strains including inbreds and F1 hybrids with relatively high success rates. Thus, cells derived from urine, which can be collected noninvasively, may be used in the rescue of endangered mammalian species by using nuclear transfer without causing injury to the animal.

Figure 1. Mouse urine-derived cells.

(A,B) Urine-derived cells derived from GFP-Tg mice. Those cells were collected from a drop of urine using a micromanipulator. Arrows show very large cells, which could not use for NT. (C) Urine-derived cell numbers for individual mice counted three times for each mouse. (D–F) Urine-derived cells were collected and stained with Hoechst stain and PI. Bright field (D), PI-positive cells (E), and Hoechst staining merged with PI staining. Arrows in (D) show dead cells, which can be distinguished without PI staining.

Figure 2. Cloned embryos and mice derived from urine-derived cell nuclei.

(A,B) Cloned morulae/blastocysts derived from GFP-Tg mouse urine-derived cells. (C–E) Immunostaining of cloned blastocysts. Cdx2-positive cells are red (C), Oct4-positive cells are green (D) and their merged image is shown in (E). (F) Cloned offspring just after caesarean section. One dead foetus was also collected. (G) Cloned males and females derived from urine-derived cell nuclei were mated with each other. About 1 month later, female clones delivered their offspring, which suggested that the clones had normal fertility.

Figure 3. Immunostaining of ntES cell line from urine-derived cells.

This ntES cell line was derived from 129GFP female mouse urine cell. AP staining (A), bright field (B), DAPI staining (C), GFP expression (D), and Oct4 immunostaining (E) of ntES cells. (B–E) are from the same sample.

Figure 4. NT using urine-derived cells.

(A) Cells were collected from urine with a micropipette and a micromanipulator. The inner diameter was about 10-15 μm. (B) The collected urine-derived cells were placed into PVP medium until use. (C,D) NT was performed using our standard method.