Supplementary Information

Fig. S1. Expression of mCherry in newborn RMCE pigletA) 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 fibroblastsA, 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 samplesA) 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 boarE) 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

Micro-satellite

Donor cells(Venus)

Piglet 1(Venus)

Piglet 2(Venus)

Piglet 3(Venus)

Piglet 4(Venus)

Piglet 5(Venus)

Piglet 6(Venus)

Piglet 7(Venus)

Piglet 8(Venus)

RMCE dc(mCherry)

Piglet 9(mCherry)

Piglet 10(mCherry)

Piglet 11(mCherry)

Piglet 12(mCherry)

Recipientgilt 1

Recipient gilt 2

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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