Hydrodynamic Delivery of Cre Protein to Lineage-Mark or Time-Stamp Mouse Hepatocytes In situ

Abstract

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.

Targeting Tanycytes: Balance between Efficiency and Specificity

“…However, some specificity may be added by adjusting the conditions of infusion. Indeed, as reported by others [43], we observed a lack of widespread Cre activity: TAT-Cre induces the DNA recombination close to the site of injection. Therefore, the flow rate, the site, and the volume of injection may influence the proportion of targeted tanycytes versus ependymal cells.…”

Dietary methionine can sustain cytosolic redox homeostasis in the mouse liver

“…Mice were kept under specialized care conditions, including continuous-flow HEPA-filtered air cages (Tecniplast); sterilized bedding and enrichment; free access to sterilized feed (Picolabs 5058) and sterilized water; and on a 14:10 light:dark cycle. For hydrodynamic delivery of Cre to liver hepatocytes, mice received a single tail-vein injection of lactated Ringer’s solution equal to 10% of their body-weight, containing 5 µg of plasmid pCMVpCreSVtx 31 . For Tamoxifen-induced conversion of all hepatocytes in mice, ROSA mT-mG/mT-mG ; Txnrd1 cond/cond ; Gsr null/null ; Alb ICE/+ mice, having an allele of the Alb ICE (for Alb - I RES- C re E RT2) knock-in allele 32 (a generous gift from P. Chambon and D. Metzger, Illkirch, France), in which an i nternal r ibosomal e ntry s ite- (IRES-) driven second cistron targeted into the 3’ untranslated region of the endogenous Alb locus was used to provide strong hepatocyte-specific transcription and translation of the Tamoxifen-inducible CreERT2 protein.…”

Cell-penetrating peptides (CPPs): From delivery of nucleic acids and antigens to transduction of engineered nucleases for application in transgenesis

“…The intracellular delivery and cytoplasmic traffic mediated by TAT and penetratin resulted in genomic recombination in subpopulations of neurons in discrete areas of embryonic and adult neural tissues (Gitton et al, 2009). TAT-Cre recombinase fusion protein was also used by Sonsteng et al (2014) to target hepatocytes in vivo by means of hydrodynamic delivery in transgenic (ROSA nTnG ) mice. The transgenic mouse version that they developed localizes the dual red-and green-fluorescent reporter proteins to the nucleus (ROSA nTnG ) instead of the outer membrane, as originally displayed in mice bearing the ROSA mTmG genotype, i.e., cells carrying the floxed membrane-targeted tdTomato (tdT) cistron followed by a membrane-targeted, enhanced green fluorescent protein (EGFP) cistron.…”