Mapping Atoh1-HA Transgene Copies

The copy number and chromosomal location of the Atoh1-HA line founder #10 [8] (link) were determined by FISH analysis. Briefly, purified plasmid (5.5 kb Atoh1-HA) DNA was labeled with red-dUTP (Alexa Fluor 594, Molecular Probes) by nick translation. The labeled transgene probe and a green-dUTP (Abbott Molecular)–labeled chromosome 10 control probe (RP24–360A19 of 245 kb bacterial artificial chromosome or BAC) were combined with sheared mouse cot DNA and hybridized to metaphase and interphase nuclei derived from the transgenic mouse lung fibroblast cultures in a solution containing 50% formamide, 10% dextran sulfate, and 2× SSC. The chromosomes were then stained with 4,6-diamidino-2-phenylindole (DAPI) and analyzed. The Atoh1-HA intensity was consistent with ∼50 copies of the transgene, as determined by genomic Southern blot analysis.

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Liu Z., Fang J., Dearman J., Zhang L, & Zuo J. (2014). In Vivo Generation of Immature Inner Hair Cells in Neonatal Mouse Cochleae by Ectopic Atoh1 Expression. PLoS ONE, 9(2), e89377.

Publication 2014

(Alexa Fluor 594 green-dUTP

Alexa 594 Bacterial artificial chromosome Chromosome 10 Chromosomes Dextran sulfate Dutp Fibroblast Fish Formamide Genomic Interphase Lung Metaphase Molecular probes Mouse Nuclei Plasmid Southern blot analysis Transgene Transgenic mouse

Corresponding Organization :

Other organizations : University of Tennessee Health Science Center, St. Jude Children's Research Hospital

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Variable analysis

independent variables

Labeled transgene probe (5.5 kb Atoh1-HA DNA labeled with red-dUTP (Alexa Fluor 594))
Chromosome 10 control probe (RP24-360A19 of 245 kb BAC labeled with green-dUTP (Abbott Molecular))

dependent variables

Copy number of the Atoh1-HA transgene
Chromosomal location of the Atoh1-HA transgene

control variables

Metaphase and interphase nuclei derived from the transgenic mouse lung fibroblast cultures
Hybridization solution containing 50% formamide, 10% dextran sulfate, and 2× SSC
Chromosomes stained with 4,6-diamidino-2-phenylindole (DAPI)

positive controls

Genomic Southern blot analysis to determine the Atoh1-HA transgene copy number (∼50 copies)

negative controls

Not explicitly mentioned

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