Cloning an Adult Jersey Cow Using Proliferating Somatic Cells

 

J.L. Edwards, F.N. Schrick, C.M. Dorado, J. Miller, L. McCann, T.J.Wilson, M. Malone, M.E. Hockett, T.M. Towns, H. Blackmon, M. Welborn and F. Hopkins

The University of Tennessee; Animal Science Department; Knoxville, TN 37996

 

Recent advancements in cloning technologies could ultimately lead to increased efficiency of animal production, discovery of genetic basis of animal and human diseases, and “molecular pharming” whereby farm animals serve as bioreactors for production of pharmaceutical proteins or organ donors. Use of quiescent-induced cell types may or may not be required for producing cloned offspring. Objective of present study was to compare development of cloned embryos reconstructed with quiescent or actively proliferating adult somatic cells. MII oocytes were enucleated between 17 and 21 h post maturation (hpm). Granulosa cells were aspirated from an adult Jersey cow using an ultrasound-guided transvaginal probe.  Primary cell lines were established and before nuclear transfer, cultured in the presence of 0.5% (serum starved; quiescent) or 10% fetal bovine serum (serum fed; proliferating). Granulosa cells were fused with recipient cytoplasts using an electrical pulse of 2.2 kV/cm for 40 :sec at 21-26 hpm. Reconstructed embryos were activated at 24-27 hpm and cultured in an atmosphere of 7% O₂ and 5.5% CO₂ in KSOMaa + BSA. Ability of cloned embryos to develop to morula or blastocyst was assessed on days 6-7 post-activation. Morulae or blastocysts were transferred to synchronized recipient heifers. Establishment of pregnancy was confirmed 29-35 post-estrus by presence of an embryonic heartbeat using ultrasound.  Development of cloned embryos to 8-16-cell by d 4 (62.6 and 59.4; SEM=6.4) and compact morula and blastocyst by d 6-7 (26.5 versus 24.9 for serum fed and starved, respectively; SEM=8.5) did not differ among treatments. Establishment of pregnancy by days 29-35 post-estrus was similar for clones reconstructed with serum fed or starved granulosa cells (Table).  Between days 30 and 60 of gestation, the majority of established pregnancies were confirmed degenerate as indicated by absence of heartbeat and detachment of placental membranes (Table).  Numerically, embryonic loss was higher in clones constructed from serum starved (88.8%) versus serum fed (36.3%) cells.  Two heifers aborted at 175 and 253 days of gestation. One heifer delivered a healthy calf at 278 days gestation.  Additional pregnancies are ongoing; one originating from serum starved versus four from serum fed cells.  Use of quiescent-induced adult cell types is not required to obtain cloned offspring. These findings should serve to hasten availability of cloning technologies to swine industry. 

 

Table:  Developmental potential of cloned embryos reconstructed with serum starved or fed adult granulosa cells after embryo transfer.

Cell Culture	Rep	Clones	M/B ET	Recips	Pregnant 29-35 d (%)	Pregnant 60 d (%)	Pregnancy Loss d 30-60 (%)
Serum Fed	6	97	27	25	11 (44.0)	7	4 (36.3)
Serum Starved	5	91	17	13	9 (69.2)	1	8 (88.8)
Total		188	44	38	20 (52.6)	8	12 (60.0)

M/B=Total number of morula and blastocysts transferred to recipient animals. 

 

Key words: nuclear transfer, cloning, somatic cells