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. 2024 Sep:9:100081.
doi: 10.1016/j.jmccpl.2024.100081. Epub 2024 Jul 3.

Comparative analysis of two independent Myh6-Cre transgenic mouse lines

Amanda Davenport  1 , Chase W Kessinger  1 , Ryan D Pfeiffer  1 , Nikita Shah  1   2 , Richard Xu  1 , E Dale Abel  3 , Nathan R Tucker  1 , Zhiqiang Lin  1
Affiliations

Comparative analysis of two independent Myh6-Cre transgenic mouse lines

Amanda Davenport et al. J Mol Cell Cardiol Plus. 2024 Sep.

Abstract

We have previously shown that the Myh6 promoter drives Cre expression in a subset of male germ line cells in three independent Myh6-Cre mouse lines, including two transgenic lines and one knock-in allele. In this study, we further compared the tissue-specificity of the two Myh6-Cre transgenic mouse lines, MDS Myh6-Cre and AUTR Myh6-Cre, through examining the expression of tdTomato (tdTom) red fluorescence protein in multiple internal organs, including the heart, brain, liver, lung, pancreas and brown adipose tissue. Our results show that MDS Myh6-Cre mainly activates tdTom reporter in the heart, whereas AUTR Myh6-Cre activates tdTom expression significantly in the heart, and in the cells of liver, pancreas and brain. In the heart, similar to MDS Myh6-Cre, AUTR Myh6-Cre activates tdTom in most cardiomyocytes. In the other organs, AUTR Myh6-Cre not only mosaically activates tdTom in some parenchymal cells, such as hepatocytes in the liver and neurons in the brain, but also turns on tdTom in some interstitial cells of unknown identity.

Keywords: Cre-loxP system; Genomic position effect; Myh6-Cre; Transgenic mouse line; tdTomato reporter.

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Figures

Fig. 1.
Fig. 1.. AUTR Myh6-Cre activates tdTom reporter in multiple internal organs.
A. Schematic representation of the targeting constructs used to generate Myh6-Cre transgenic mouse lines. Numbered black bars indicate Myh6 gene exons. Dotted lines outline the PCR amplicons used for genotyping. B. PCR-based genotyping. Mouse Tail DNA was used as template. C. Breeding scheme that leads to the production of AUTR Myh6-Cre;tdTom and MDS Myh6-Cre; tdTom. D–F. Ex vivo fluorescent imaging of mouse organs. 2-month old mice were included in this study. Fluorescent imaging was performed with Perkin Elmer IVIS Spectrum system. Colour range indicates radiant efficiency ([p/s/cm2/sr]/[μW/cm2]).
Fig. 2.
Fig. 2.. AUTR Myh6-Cre depletes the stop cassette of the tdTom transgenic construct in multiple organs.
A. Depiction of the PCR strategy for detecting Stop cassette depletion. Pr-1 and Pr-2 primer pair was used to detect the tdTom DNA sequence. Pr-3 and Pr-4 primer pair was used to examine the occurrence of Cre-mediated Stop cassette depletion. B–D. PCR results. Genomic DNA from different organs was used as PCR template. H, heart; L, liver, P, Pancreas. Red stars indicate the PCR amplicons derived from the recombined tdTom reporter allele.
Fig. 3.
Fig. 3.. AUTR Myh6-Cre activates tdTom in cardiomyocytes (CMs) and other cell types. A-B.
tdTom fluorescence images of the adult heart (A) and liver (B) sections. P42, postnatal day 42. B, yellow arrows indicate hepatocytes; yellow stars indicate non-hepatocytes. C. tdTom and AUTR Myh6-Cre;tdTom heart sections at embyronic stage 18.5 (E18.5). White arrows indicate non-CMs. Two myocardial regions from the same heart are presented. D. tdTom and AUTR Myh6-Cre;tdTom liver sections at P14. Two regions from the same liver section are presented. A, B, C and D, scale bar = 50 μm.
Fig. 4.
Fig. 4.. AUTR Myh6-Cre activates tdTom reporter in the brain.
A. Ex vivo fluorescent imaging of brain. 2-month old mice were included in this study. Colour range indicates radiant efficiency ([p/s/cm2/sr]/[μW/cm2]). B–C. tdTom fluorescence images of the sagital brain sections. B, tdTom signal distribution in the whole brain. CC, corpus callosum; SC, superior colliculus. Scale bar = 1000 μm. C, magnified images of tdTom positive cells in different brain regions. Scale bar = 50 μm.
Fig. 5.
Fig. 5.. AUTR Myh6-Cre activates tdTom reporter in both neurons and non-neuron cells.
Confocal images of olfactory bulb (A), hippocampus dentate gyrus (B), isocortex (C) and cerebellar cortex (D). NeuN antibody was used to label the cell bodies of mature neurons. White arrows indicate tdTom positive mature neurons, and yellow arrowheads indicate non-neuron cells. A, MOB: main olfactory bulb; OP; olfactory peduncle. B, ml: molecular layer; gcl: granule cell layer; pl: polymorphic layer (pl). In panel D, P indicates Purkinje cells. Scale bar = 50 μm.
Fig. 6.
Fig. 6.. Identification of the AUTR Myh6-Cre transgenic DNA insertion locus.
A. The chromosomes enriched with TLA signals. B. Zoom-in view of the AUTR Myh76-Cre and MDS Myh6-Cre TLA peaks. C. Genomic loci carrying the integrated transgenic DNA cassettes.
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