nature | methods Lifeact mice for studying F-actin dynamics Julia Riedl, Kevin C Flynn, Aurelia Raducanu, Florian Gärtner, Gisela Beck, Michael Bösl, Frank Bradke, Steffen Massberg, Attila Aszodi, Michael Sixt & Roland Wedlich-Söldner Supplementary figures and text: Supplementary Figure 1 Construct generated for pronuclear injection. Supplementary Figure 2 Lifeact-EGFP and -mRFPruby are expressed during embryonic development. Supplementary Figure 3 Lifeact-EGFP and -mRFPruby expression in organs. Supplementary Figure 4 Tissue labeling of Lifeact-EGFP mice. Supplementary Figure 5 Tissue labeling of Lifeact-mRFPruby mice. Supplementary Figure 6 Lymphocytes express Lifeact-EGFP at high levels. Supplementary Methods Description of techniques used for manipulation, isolation and imaging of whole mice, mice tissues and cultured primary cells. Supplementary Video 1 Migrating T-cell expressing Lifeact-EGFP. Images were acquired by TIRFM. Scale bar: 3 μm. Supplementary Video 2 Spreading platelet expressing Lifeact-EGFP. Images were acquired by TIRFM. Scale bar: 3 μm. Supplementary Video 3 Dividing chondrocytes. Images of tibial cartilage slice cultures were acquired on a spinning disk microscope. Scale bar: 20 μm. Note: Supplementary Videos 1–3 are available on the Nature Methods website. Nature Methods, vol. 7, no. 3 Wedlich-Soldner, R. et al.
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nature methods · Stomach Uterus Skeletal muscle Heart Alexa 560 phalloidin Lifeact-EGFP Overlay Overlay Alexa 560 phalloidin Lifeact- ... was added overnight and the following day
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nature | methods
Lifeact mice for studying F-actin dynamics Julia Riedl, Kevin C Flynn, Aurelia Raducanu, Florian Gärtner, Gisela Beck, Michael Bösl, Frank Bradke, Steffen Massberg, Attila Aszodi, Michael Sixt & Roland Wedlich-Söldner
Supplementary figures and text:
Supplementary Figure 1 Construct generated for pronuclear injection.
Supplementary Figure 2 Lifeact-EGFP and -mRFPruby are expressed during embryonic development.
Supplementary Figure 3 Lifeact-EGFP and -mRFPruby expression in organs.
Supplementary Figure 4 Tissue labeling of Lifeact-EGFP mice.
Supplementary Figure 5 Tissue labeling of Lifeact-mRFPruby mice.
Supplementary Figure 6 Lymphocytes express Lifeact-EGFP at high levels.
Supplementary Methods Description of techniques used for manipulation, isolation and
imaging of whole mice, mice tissues and cultured primary cells.
Supplementary Video 1 Migrating T-cell expressing Lifeact-EGFP. Images were acquired by TIRFM. Scale bar: 3 µm.
Supplementary Video 2 Spreading platelet expressing Lifeact-EGFP. Images were acquired by TIRFM. Scale bar: 3 µm.
Supplementary Video 3 Dividing chondrocytes. Images of tibial cartilage slice cultures were acquired on a spinning disk microscope. Scale bar: 20 µm.
Note: Supplementary Videos 1–3 are available on the Nature Methods website.
Nature Methods, vol. 7, no. 3 Wedlich-Soldner, R. et al.
Heart Brain Muscle Kidney Liver Spleen
E4.5
a
E10.5
E10.5
b
Supplementary Figure 1. Construct generated for pronuclear injection.
Supplementary Figure 2. Lifeact-EGFP and -mRFPruby are expressed during embryonic development.
(a) Fertilized oocytes were isolated and cultured in vitro till E4.5. Images were acquired by epifluorescence. Scale bar: 20 μm. (b) Embryos were dissected at E10.5 and imaged with a stereo microscope. Scale bar: 1 mm.
Supplementary Figure 3. Lifeact-EGFP and -mRFPruby expression in organs.
After linearization of the vector the indicated fragment containing Lifeact-EGFP or –mRFPruby was purified and used for injection. The sequence contains the chicken beta-actin promoter coupled to a CMV enhancer and intron (promoter, dark grey) in front of the Lifeact-EGFP or -mRFPruby sequence (purple-green/red) and a Poly-A sequence (light grey).
Heart, brain, skeletal muscle from femur, kidney, liver and spleen were isolated from transgenic mice and imaged with a stereo microscope.
Nature Methods, vol. 7, no. 3 Wedlich-Soldner, R. et al.
Large intestine
Small intestine
Brain
Liver
Lymphnode
Spleen
Lung
Kidney
Thyroid gland
Thymus
Stomach Uterus
Skeletal muscle Heart
Alexa 560phalloidin
Lifeact-EGFP Overlay Overlay
Alexa 560phalloidin
Lifeact-EGFP
Supplementary Figure 4. Tissue labeling of Lifeact-EGFP mice.
Colocalization of GFP signals with F-actin structures labeled by Alexa 560-phalloidin is shown. Cryosections of 14 organs were counterstained with phalloidin. Scale bar: 50 μm.
Nature Methods, vol. 7, no. 3 Wedlich-Soldner, R. et al.
Supplementary Figure 5. Tissue labeling of Lifeact-mRFPruby mice.
Large intestine
Small intestine
Brain
Liver
Lymphnode
Spleen
Lung
Kidney
Thyroid gland
Thymus
Stomach Uterus
Skeletal muscle Heart
Alexa 488phalloidin
Lifeact-mRFPruby
Alexa 488phalloidin
Lifeact-mRFPrubyOverlay Overlay
Colocalization of mRFPruby signals with F-actin structures labeled by Alexa 488-phalloidin is shown. Cryosections of 14 organs were counterstained with phalloidin. Scale bar: 50 μm.
Nature Methods, vol. 7, no. 3 Wedlich-Soldner, R. et al.
CD8+ T cellsTotal splenocytes
fluorescencefluorescence
coun
t
coun
t
Supplementary Figure 6. Lymphocytes express Lifeact-EGFP at high levels.
Flow cytometric analysis of GFP-fluorescence of total splenocytes and CD8-positive T-cells (wt control: grey shaded; Lifeact-EGFP splenocytes: black line, unshaded)
Nature Methods, vol. 7, no. 3 Wedlich-Soldner, R. et al.
Supplementary Methods
Generation of transgenic mice
To obtain transgenic mice we generated two constructs, originating from pCAG-
vector1, with a cytomegalovirus enhancer, chicken-�-actin promoter, a chimeric
intron followed either by the Lifeact-EGFP or the Lifeact-mRFPruby2 sequence and a
poly(A)-tail. Constructs were digested with AccI and HindIII (EGFP) and AccI-PstI
(mRFPruby), the linearized DNA was injected into fertilized oocytes (C57BL6/N x
FVB/N (F2)) and transferred into pseudo-pregnant females. The insertion of either
transgene into the genome was tested in >100 pups of each strain by PCR (primers