Cloned ferrets produced by somatic cell nuclear transfer

Abstract

Somatic cell nuclear transfer (SCNT) offers great potential for developing better animal models of human disease. The domestic ferret (Mustela putorius furo) is an ideal animal model for influenza infections and potentially other human respiratory diseases such as cystic fibrosis, where mouse models have failed to reproduce the human disease phenotype. Here, we report the successful production of live cloned, reproductively competent, ferrets using species-specific SCNT methodologies. Critical to developing a successful SCNT protocol for the ferret was the finding that hormonal treatment, normally used for superovulation, adversely affected the developmental potential of recipient oocytes. The onset of Oct4 expression was delayed and incomplete in parthenogenetically activated oocytes collected from hormone-treated females relative to oocytes collected from females naturally mated with vasectomized males. Stimulation induced by mating and in vitro oocyte maturation produced the optimal oocyte recipient for SCNT. Although nuclear injection and cell fusion produced mid-term fetuses at equivalent rates (approximately 3-4%), only cell fusion gave rise to healthy surviving clones. Single cell fusion rates and the efficiency of SCNT were also enhanced by placing two somatic cells into the perivitelline space. These species-specific modifications facilitated the birth of live, healthy, and fertile cloned ferrets. The development of microsatellite genotyping for domestic ferrets confirmed that ferret clones were genetically derived from their respective somatic cells and unrelated to their surrogate mother. With this technology, it is now feasible to begin generating genetically defined ferrets for studying transmissible and inherited human lung diseases. Cloning of the domestic ferret may also aid in recovery and conservation of the endangered black-footed ferret and European mink.

Fig. 1

In vivo development of 21-day fetuses following SCNT. Gross anatomical examples of implantations and dissected fetuses at 21 days for fibroblast and cumulous clones as compared to natural mating with fertile males.

Fig. 2

(A) Two cloned sable coat-color female ferret pups, Libby and Lilly (at 7 weeks of age), produced by SCNT and ET into an albino surrogate Jill. (B) Lilly with her 6 newborn pups. (C) Libby with her 10 newborn pups.

Fig. 3

In vitro development of fibroblast NT embryos following cell fusion and activation. (A) DAPI staining of PCC formation at 2 h post-SCNT. (B) DAPI staining of pronuclei formation at 4 h post-SCNT. (C) Bright field photomicrograph of 7-day blastocysts formed following SCNT. (D) DAPI stained 7-day blastocysts formed following SCNT.

Fig. 4

Oct4 staining of parthenogenetically activated oocytes. Oocytes were collected (A) following eCG treatment of donor Jills or (B) following natural mating with vasectomized males. Oocytes were activated as described in Materials and methods and stained with anti-Oct4 antibodies and DAPI at various stages as indicated. A total of four Jills were used to generate at least 6 embryos in each developmental group. Oct4 expression was seen in 47 ± 14% of nuclei from ≤21 cells embryos of the hormonally treated group and 83 ± 5% of nuclei from ≤21 cells embryos in the untreated group.