5-Animals: Cattle cloned from adult ear fibroblasts

[GENETNL <genetnl@xs4all.be>]

----------------------------- GENET-news -----------------------------

TITLE:  Cattle cloned from adult ear fibroblasts
SOURCE: ISB News Report - March 2000
        by Eric. A. Wong
DATE:   March 3, 2000

-------------------- archive: http://www.gene.ch/ --------------------


Cattle cloned from adult ear fibroblasts

The ability to precisely modify the mouse genome has been facilitated 
by the isolation and manipulation of embryonic stem (ES) cells. ES 
cells can be maintained in a totipotent or undifferentiated state 
during long-term culture while selection for cells with targeted 
genetic modifications is performed. Transgenic livestock with 
targeted genetic modifications cannot be generated by this approach 
because livestock ES cells comparable to mouse ES cells have not yet 
been isolated. An alternate approach for performing precise genetic 
modifications in livestock lies in the application of nuclear 
transfer technology, i.e., cloning. In this technique, cells can be 
cultured and used as nuclear donors. Therefore, a key issue is 
whether cells can be maintained in culture for a prolonged period in 
order to allow time for selection of cells with precise genetic 
modifications, and still serve as nuclear donors.

In the February 1, 2000 issue of the Proceedings of the National 
Academy of Sciences (USA), researchers at the Kagoshima Prefectural 
Cattle Breeding Institute of Japan and the University of Connecticut 
report the production of six cloned calves from adult fibroblast 
cells after long-term culture. Skin fibroblasts were collected from 
the ear of a 17-year old Japanese Black Beef bull and cultured for up 
to three months prior to nuclear transfer.

The physiological status and passage number of the cultured ear 
fibroblasts affected the efficiency of cloning. As had been 
previously reported in sheep cloning studies, serum starvation prior 
to nuclear transfer is an important step. No calves were born when 
nuclei were obtained from non-serum starved cells. Higher development 
rates for embryos were observed when nuclei were obtained from cells 
maintained in culture for 10-15 passages compared with cells 
maintained for five passages.

>From 54 manipulated embryos, six live calves were born. Three 
pregnancies were established using passage five cells, but all three 
fetuses were aborted. Nine pregnancies were established using passage 
10 cells and four live bull calves were born. Two of these four 
calves died shortly after birth. Using passage 15 cells, two out of 
three pregnancies resulted in live bull calves.

The authors speculate that the reason for the reduced cloning 
efficiency using passage five cells is due to the heterogeneity of 
the cell population. At passage five there was a mix of fibroblasts 
and other cell types, such as epithelial cells, which are inferior to 
fibroblasts for cloning. At later passages the more efficient 
fibroblast cells outgrew the epithelial cells.

As had been seen in previous studies, the gestation periods of the 
cloned calves were longer and the birth weights were larger. The 
gestation periods for the cloned calves were on average nine days 
longer than the average gestation period for the breed. The birth 
weights of the clones were 20% heavier than the average birth weight. 
However, at the time of writing, the four surviving bull calves were 
10-12 months of age, healthy, and normal.

These results demonstrate that adult fibroblast cells can be 
maintained in long-term culture without loss of competency to form 
clones by nuclear transfer. Cells were cultured for up to three 
months, which represented up to 15 passages and 45 cell doublings. 
This is sufficient time to allow the selection of cells with targeted 
genetic modifications. The next step will be to determine if selected 
cells also maintain competency to serve as nuclear donors. If so, 
then nuclear transfer can be used to circumvent the current 
limitation of the lack of ES cells to generate livestock with precise 
genetic modifications. 
Source
Kubota C, et al. 2000. Six cloned calves produced from adult 
fibroblast cells after long-term culture. Proceedings of the National 
Academy of Science USA 97: 990-995.

Eric A. Wong
Department of Animal and Poultry Sciences
Virginia Tech
ewong@vt.edu

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