Isolation of Cardiomyocytes from Alms1 Gt/Gt Mice

The Alms1^Gt/Gt mice (B6.129P2-Alms1^Gt(XH152)Byg/Pjn) used in this study were generated from gene-trapped ES cells (MMRC#008633)^{26 (link),47 (link)}. Mice were fed ad libitum a 4K54 diet (PMI Nutrition International, St. Louis, MO) and provided an unlimited access to water in a temperature/humidity controlled setting with a 12 hour light/dark cycle at The Jackson Laboratory Research Animal Facility. All mouse protocols used in this study were approved by the JAX institutional Animal Care and Use Committee. Mouse hearts were extracted from mice euthanized by carbon dioxide asphyxiation and allowed to contract in phosphate buffered saline for 5–10 minutes and immediately placed in 4% paraformaldeyde at 4 °C overnight. Subsequently, tissues were embedded in paraffin, sectioned, and immunostained as previously reported^{48 (link)}. The aMHC promoter-driven EGFP-IRES-puromycin transgenic mice (alphaMHC-GFP), in which only cardiomyocytes express the green fluorescent protein (GFP) facilitate rapid and efficient isolation of cardiomyocytes^{23 (link)}. Hearts from these mice were dissociated using collagense II/TrypLE. GFP+ cardiomyocytes were isolated by Fluorescence Activated Cell Sorting (FACS) using an SH800 sorter (Sony Biotechnology).

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Shenje L.T., Andersen P., Halushka M.K., Lui C., Fernandez L., Collin G.B., Amat-Alarcon N., Meschino W., Cutz E., Chang K., Yonescu R., Batista D.A., Chen Y., Chelko S., Crosson J.E., Scheel J., Vricella L., Craig B.D., Marosy B.A., Mohr D.W., Hetrick K.N., Romm J.M., Scott A.F., Valle D., Naggert J.K., Kwon C., Doheny K.F, & Judge D.P. (2014). Mutations in Alström Protein Impair Terminal Differentiation of Cardiomyocytes. Nature communications, 5, 3416.

Publication 2014

4K54 diet SH800 sorter

Animal Asphyxiation Carbon dioxide Cardiomyocytes Diet Embedded paraffin Es cells Gene Green fluorescent protein Hearts Humidity Institutional animal care and use committee Ires Isolation Laboratory animal Mice Phosphate Puromycin Saline Tissues Transgenic mice

Corresponding Organization :

Other organizations : Johns Hopkins Medicine, Johns Hopkins University, Jackson Laboratory, North York General Hospital, Hospital for Sick Children, National University of Singapore

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Variable analysis

independent variables

Mouse genotype: Alms1^Gt/Gt (B6.129P2-Alms1^Gt(XH152)Byg/Pjn)
Mouse heart dissociation and cardiomyocyte isolation: Collagenase II/TrypLE treatment, Fluorescence Activated Cell Sorting (FACS) of GFP+ cardiomyocytes from alphaMHC-GFP transgenic mice

dependent variables

Cardiomyocyte morphology and function (not explicitly mentioned)

control variables

Mouse diet: 4K54 diet (PMI Nutrition International, St. Louis, MO)
Mouse housing: Temperature/humidity controlled setting with 12-hour light/dark cycle
Mouse euthanasia method: Carbon dioxide asphyxiation
Mouse heart tissue processing: Fixation in 4% paraformaldehyde, paraffin embedding, sectioning, and immunostaining

positive controls

AlphaMHC-GFP transgenic mice, in which only cardiomyocytes express the green fluorescent protein (GFP), for efficient isolation of cardiomyocytes

negative controls

Not explicitly mentioned

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