Hydrodynamic delivery of Cre protein to lineage-mark or time-stamp mouse hepatocytes in situ

Katherine M Sonsteng; Justin R Prigge; Emily A Talago; Ronald K June; Edward E Schmidt

doi:10.1371/journal.pone.0091219

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Hydrodynamic delivery of Cre protein to lineage-mark or time-stamp mouse hepatocytes in situ

Journal article

Open access

Hydrodynamic delivery of Cre protein to lineage-mark or time-stamp mouse hepatocytes in situ

Katherine M Sonsteng, Justin R Prigge, Emily A Talago, Ronald K June and Edward E Schmidt

PloS one, Vol.9(3), pp.e91219-e91219

2014

DOI: https://doi.org/10.1371/journal.pone.0091219

Handle:

https://hdl.handle.net/2376/100534

PMCID: PMC3953374

PMID: 24626158

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Abstract

Recombinant Proteins - metabolism

Fluorescent Dyes - chemistry

Green Fluorescent Proteins - metabolism

Liver - metabolism

Hydrodynamics

Genetic Markers

Hepatocytes - metabolism

Plasmids - metabolism

Cell Lineage

Hepatocytes - cytology

Animals

Alleles

Integrases - metabolism

Mice

Cell-Penetrating Peptides - metabolism

Crosses, Genetic

tat Gene Products, Human Immunodeficiency Virus - metabolism

Cre-responsive fluorescent marker alleles are powerful tools for cell lineage tracing in mice; however their utility is limited by regulation of Cre activity. When targeting hepatocytes, hydrodynamic delivery of a Cre-expression plasmid can convert Cre-responsive alleles without inducing the intracellular or systemic antiviral responses often associated with viral-derived Cre-expression vectors. In this method, rapid high-volume intravenous inoculation induces hepatocyte-targeted uptake of extracellular molecules. Here we tested whether hydrodynamic delivery of Cre protein or Cre fused to the HIV-TAT cell-penetrating peptide could convert Cre-responsive reporters in hepatocytes of mice. Hydrodynamic delivery of 2 nmol of either Cre or TAT-Cre protein converted the reporter allele in 5 to 20% of hepatocytes. Neither protein gave detectable Cre activity in endothelia, non-liver organs, or non-hepatocyte cells in liver. Using mice homozygous for a Cre-responsive marker that directs red- (Cre-naïve) or green- (Cre-converted) fluorescent proteins to the nucleus, we assessed sub-saturation Cre-activity. One month after hydrodynamic inoculation with Cre protein, 58% of hepatocyte nuclei that were green were also red, indicating that less than half of the hepatocytes that had obtained enough Cre to convert one marker allele to green were able to convert all alleles. For comparison, one month after hydrodynamic delivery of a Cre-expression plasmid with a weak promoter, only 26% of the green nuclei were also red. Our results show that hydrodynamic delivery of Cre protein allows rapid allelic conversion in hepatocytes, but Cre-activity is sub-saturating so many cells will not convert multiple Cre-responsive alleles.

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Title: Hydrodynamic delivery of Cre protein to lineage-mark or time-stamp mouse hepatocytes in situ
Creators: Katherine M Sonsteng - Department of Immunology and Infectious Diseases, Montana State University, Bozeman, Montana, United States of America
Justin R Prigge - Department of Immunology and Infectious Diseases, Montana State University, Bozeman, Montana, United States of America
Emily A Talago - Department of Immunology and Infectious Diseases, Montana State University, Bozeman, Montana, United States of America
Ronald K June - Department of Mechanical and Industrial Engineering, Montana State University, Bozeman, Montana, United States of America
Edward E Schmidt - Department of Immunology and Infectious Diseases, Montana State University, Bozeman, Montana, United States of America
Publication Details: PloS one, Vol.9(3), pp.e91219-e91219
Academic Unit: UNKNOWN
Publisher: United States
Grant note: GM103500 / NIGMS NIH HHS R01 AG040020 / NIA NIH HHS GM103394 / NIGMS NIH HHS AG040020 / NIA NIH HHS P20 GM103394 / NIGMS NIH HHS P20 GM103500 / NIGMS NIH HHS
Identifiers: 99900546527101842
Language: English
Resource Type: Journal article