Supplementary Information
Fig. S1. Expression of mCherry in newborn RMCE piglet
A) Organ samples of a stillborn piglet derived from RMCE/SCNT (brightfield conditions).
B) Same view under specific excitation of mCherry. Note, the ubiquitous organ-specific
expression of mCherry. The high expression of mCherry in muscle (skeletal muscle and
heart) and skin, and moderate to low expression levels in kidney, lung and liver are
comparable to the respective expression of Venus in the donor animals (Garrels et al.,
2011)
C) Expression of mCherry in leukocytes (red), but not in erythrocytes.
D) Overlay of direct fluorescence and brightfield view, some erythrocytes are labelled by
arrows.
E) Brightfield view of D.
F) Flow cytometric analysis of non-transgenic porcine fibroblasts.
G) Flow cytometric analysis of Venus-transgenic fibroblasts.
H) Flow cytometric analysis of mCherry-transgenic fibroblasts.
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Supplementary Fig.S2. Expression in cultured fibroblasts
A, B) Representative images of specific fluorescence in fibroblasts derived from Venus (A) or
mCherry pigs (B).
C, D) Corresponding bright field images, nuclei are counterstained with Hoechst33342.
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Supplementary Fig.S3. Quantification of mCherry and Venus expression in porcine organ
samples
A) Western blot of a dilution series of recombinant mCherry protein (63 to 1000 ng), loaded
in parallel to protein extracts from a control pig (wt) or organs from a mCherry animal
(kidney, liver, heart) (10 g each). Arrows indicate the position of the recombinant
mCherry (~ 35 kD). Note, that the recombinant, but also the organ samples showed faster
migrating (degradation) products, which are recognized by the polyclonal antibody.
B) Corresponding Coomassie-stained gel.
C) Western blot of a GFP fusion protein of 58 kD (63 and 125 ng) and protein extracts from
organs of a Venus pig and a wildtype pig (wt). 10 and 2.5 g of the organ samples from
the Venus pig were loaded. Arrows indicate the position of the 58 kD fusion protein.
D) Corresponding Coomassie-stained gel.
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Supplementary Fig.S4. Germ line transmission of a mCherry boar
E) PCR genotyping of a litter of 9 piglets (1-9) derived from insemination of a wildtype sow
with semen of one of the mCherry boars. Piglets 1-5 were positive for a mCherry
amplicon. M, DNA size marker; –c, wildtype control; +c, positive control (mCherry boar).
F) Expression of mCherry protein in offspring. Proteins were extracted from skin biopsies
and probed with an anti-mCherry antibody. M, Magic Mark size marker; 1-9, offspring
samples.
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Supplementary Table S1
Microsatellite analysis of cell cultures, piglets and foster mothers
Donor cells Piglet 1
(Venus)
(Venus)
Piglet 2
(Venus)
Piglet 3
(Venus)
Piglet 4
(Venus)
Piglet 5
(Venus)
Piglet 6
(Venus)
Piglet 7
(Venus)
Piglet 8
(Venus)
RMCE dc Piglet 9
(mCherry) (mCherry)
Piglet 10
(mCherry)
Piglet 11
(mCherry)
Piglet 12 Recipient
(mCherry) gilt 1
Recipient
gilt 2
Microsatellite
S0002
208/206
208/206
206/206
208/206
208/206
208/206
208/206
208/206
208/206
208/206
208/206
208/206
208/206
208/206
206/198
208/206
S0101
214/212
214/212
214/212
214/212
214/212
214/212
214/212
214/212
214/212
214/212
214/212
214/212
214/212
214/212
212/212
212/210
SW122
122/116
122/116
122/116
122/116
122/116
122/116
122/116
122/116
122/116
122/116
122/116
122/116
122/116
122/116
122/112
116/112
SW632
171/165
171/165
171/165
171/165
171/165
171/165
171/165
171/165
171/165
171/165
171/165
171/165
171/165
171/165
173/173
171/165
SW951
123/123
123/123
123/123
123/123
123/123
123/123
123/123
123/123
123/123
123/123
123/123
123/123
123/123
123/123
125/125
125/125
SW857
105/94
105/94
105/94
105/94
105/94
105/94
105/94
105/94
105/94
105/94
105/94
105/94
105/94
105/94
110/94
105/94
SW911
Np
Np
Np
Np
Np
Np
Np
Np
Np
Np
Np
Np
Np
Np
Np
Np
Numbering of piglets according to Fig.4; RMCE dc, RMCE donor cells; Np, non polymorphic.
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