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-na？ve) 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.
References
[1]
Soriano P (1999) Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 21: 70–71.
[2]
Muzumdar MD, Tasic B, Miyamichi K, Li L, Luo L (2007) A global double-fluorescent Cre reporter mouse. Genesis 45: 593–605. doi: 10.1002/dvg.20335
[3]
Prigge JR, Wiley JA, Talago EA, Young EM, Johns LL, et al. (2013) Nuclear double-fluorescent reporter for in vivo and ex vivo analyses of biological transitions in mouse nuclei. Mamm Genome. 24: 389–399. doi: 10.1007/s00335-013-9469-8
[4]
Hans S, Freudenreich D, Geffarth M, Kaslin J, Machate A, et al. (2011) Generation of a non-leaky heat shock-inducible Cre line for conditional Cre/lox strategies in zebrafish. Dev Dyn 240: 108–115. doi: 10.1002/dvdy.22497
[5]
Luche H, Weber O, Nageswara Rao T, Blum C, Fehling HJ (2007) Faithful activation of an extra-bright red fluorescent protein in “knock-in” Cre-reporter mice ideally suited for lineage tracing studies. Eur J Immunol 37: 43–53. doi: 10.1002/eji.200636745
[6]
Brockschnieder D, Pechmann Y, Sonnenberg-Riethmacher E, Riethmacher D (2006) An improved mouse line for Cre-induced cell ablation due to diphtheria toxin A, expressed from the Rosa26 locus. Genesis 44: 322–327. doi: 10.1002/dvg.20218
[7]
Mahonen AJ, Airenne KJ, Lind MM, Lesch HP, Yla-Herttuala S (2004) Optimized self-excising Cre-expression cassette for mammalian cells. Biochem Biophys Res Commun 320: 366–371. doi: 10.1016/j.bbrc.2004.05.175
[8]
Mack A, Sauer B, Abremski K, Hoess R (1992) Stoichiometry of the Cre recombinase bound to the lox recombining site. Nucleic Acids Res 20: 4451–4455. doi: 10.1093/nar/20.17.4451
[9]
Schmidt EE, Taylor DS, Prigge JR, Barnett S, Capecchi MR (2000) Illegitimate Cre-dependent chromosome rearrangements in transgenic mouse spermatids. Proc Natl Acad Sci U S A 97: 13702–13707. doi: 10.1073/pnas.240471297
[10]
Shi J, Petrie HT (2012) Activation kinetics and off-target effects of thymus-initiated cre transgenes. PLoS ONE 7: e46590. doi: 10.1371/journal.pone.0046590
[11]
Overturf K, al-Dhalimy M, Ou CN, Finegold M, Grompe M (1997) Serial transplantation reveals the stem-cell-like regenerative potential of adult mouse hepatocytes. Am J Pathol 151: 1273–1280.
[12]
Grompe M, Laconi E, Shafritz DA (1999) Principles of therapeutic liver repopulation. Semin Liver Dis 19: 7–14. doi: 10.1055/s-2007-1007093
[13]
Badea TC, Wang Y, Nathans J (2003) A noninvasive genetic/pharmacologic strategy for visualizing cell morphology and clonal relationships in the mouse. J Neurosci 23: 2314–2322.
[14]
Schuler M, Dierich A, Chambon P, Metzger D (2004) Efficient temporally controlled targeted somatic mutagenesis in hepatocytes of the mouse. Genesis 39: 167–172. doi: 10.1002/gene.20039
[15]
Suvorova ES, Lucas O, Weisend CM, Rollins MF, Merrill GF, et al. (2009) Cytoprotective Nrf2 pathway is induced in chronically txnrd 1-deficient hepatocytes. PLoS One 4: e6158. doi: 10.1371/journal.pone.0006158
[16]
Malato Y, Naqvi S, Schurmann N, Ng R, Wang B, et al. (2011) Fate tracing of mature hepatocytes in mouse liver homeostasis and regeneration. J Clin Invest 121: 4850–4860. doi: 10.1172/jci59261
[17]
Jayandharan GR, Aslanidi G, Martino AT, Jahn SC, Perrin GQ, et al. (2011) Activation of the NF-kappaB pathway by adeno-associated virus (AAV) vectors and its implications in immune response and gene therapy. Proc Natl Acad Sci U S A 108: 3743–3748. doi: 10.1073/pnas.1012753108
[18]
Kimura T, Koya RC, Anselmi L, Sternini C, Wang HJ, et al. (2007) Lentiviral vectors with CMV or MHCII promoters administered in vivo: immune reactivity versus persistence of expression. Mol Ther 15: 1390–1399. doi: 10.1038/sj.mt.6300180
[19]
Benihoud K, Salone B, Esselin S, Opolon P, Poli V, et al. (2000) The role of IL-6 in the inflammatory and humoral response to adenoviral vectors. J Gene Med 2: 194–203. doi: 10.1002/(sici)1521-2254(200005/06)2:3<194::aid-jgm102>3.0.co;2-5
[20]
Christ M, Louis B, Stoeckel F, Dieterle A, Grave L, et al. (2000) Modulation of the inflammatory properties and hepatotoxicity of recombinant adenovirus vectors by the viral E4 gene products. Hum Gene Ther 11: 415–427. doi: 10.1089/10430340050015888
[21]
Muruve DA, Barnes MJ, Stillman IE, Libermann TA (1999) Adenoviral gene therapy leads to rapid induction of multiple chemokines and acute neutrophil-dependent hepatic injury in vivo. Hum Gene Ther 10: 965–976. doi: 10.1089/10430349950018364
[22]
Zhu HZ, Wang W, Feng DM, Sai Y, Xue JL (2006) Conditional gene modification in mouse liver using hydrodynamic delivery of plasmid DNA encoding Cre recombinase. FEBS Lett 580: 4346–4352. doi: 10.1016/j.febslet.2006.06.094
[23]
Keng VW, Villanueva A, Chiang DY, Dupuy AJ, Ryan BJ, et al. (2009) A conditional transposon-based insertional mutagenesis screen for genes associated with mouse hepatocellular carcinoma. Nat Biotechnol 27: 264–274. doi: 10.1038/nbt.1526
[24]
Sebestyen MG, Budker VG, Budker T, Subbotin VM, Zhang G, et al. (2006) Mechanism of plasmid delivery by hydrodynamic tail vein injection. I. Hepatocyte uptake of various molecules. J Gene Med 8: 852–873. doi: 10.1002/jgm.921
[25]
Keng VW, Tschida BR, Bell JB, Largaespada DA (2011) Modeling hepatitis B virus X-induced hepatocellular carcinoma in mice with the Sleeping Beauty transposon system. Hepatology 53: 781–790. doi: 10.1002/hep.24091
[26]
Gump JM, June RK, Dowdy SF (2010) Revised role of glycosaminoglycans in TAT protein transduction domain-mediated cellular transduction. J Biol Chem 285: 1500–1507. doi: 10.1074/jbc.m109.021964
[27]
Wadia JS, Stan RV, Dowdy SF (2004) Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nat Med 10: 310–315. doi: 10.1038/nm996
[28]
Thomas KR, Capecchi MR (1987) Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51: 503–512. doi: 10.1016/0092-8674(87)90646-5
[29]
June RK, Gogoi K, Eguchi A, Cui XS, Dowdy SF (2010) Synthesis of a pH-sensitive nitrilotriacetic linker to peptide transduction domains to enable intracellular delivery of histidine imidazole ring-containing macromolecules. J Am Chem Soc 132: 10680–10682. doi: 10.1021/ja1040418
[30]
Rollins MF, van der Heide DM, Weisend CM, Kundert JA, Comstock KM, et al. (2010) Hepatocytes lacking thioredoxin reductase 1 have normal replicative potential during development and regeneration. J Cell Sci 123: 2402–2412. doi: 10.1242/jcs.068106
[31]
Iverson SV, Comstock KM, Kundert JA, Schmidt EE (2011) Contributions of new hepatocyte lineages to liver growth, maintenance, and regeneration in mice. Hepatology 54: 655–663. doi: 10.1002/hep.24398
[32]
Weisend CM, Kundert JA, Suvorova ES, Prigge JR, Schmidt EE (2009) Cre activity in fetal albCre mouse hepatocytes: Utility for developmental studies. Genesis 47: 789–792. doi: 10.1002/dvg.20568
[33]
Jutila MA, Berg EL, Kroese FG, Rott L, Perry V, et al. (1993) In vivo distribution and characterization of two novel mononuclear phagocyte differentiation antigens in mice. J Leukoc Biol 54: 30–39.
[34]
Buffone A Jr, Mondal N, Gupta R, McHugh KP, Lau JT, et al. (2013) Silencing alpha1,3-fucosyltransferases in human leukocytes reveals a role for FUT9 enzyme during E-selectin-mediated cell adhesion. J Biol Chem 288: 1620–1633. doi: 10.1074/jbc.m112.400929
[35]
Huang M, Sun R, Wei H, Tian Z (2013) Simultaneous knockdown of multiple ligands of innate receptor NKG2D prevents natural killer cell-mediated fulminant hepatitis in mice. Hepatology 57: 277–288. doi: 10.1002/hep.25959
[36]
Shigekawa M, Hikita H, Kodama T, Shimizu S, Li W, et al. (2012) Pancreatic STAT3 protects mice against caerulein-induced pancreatitis via PAP1 induction. Am J Pathol 181: 2105–2113. doi: 10.1016/j.ajpath.2012.08.038
[37]
Zhou XJ, Sun SH, Wang P, Yu H, Hu JY, et al. (2012) Over-expression of uPA increases risk of liver injury in pAAV-HBV transfected mice. World J Gastroenterol 18: 1892–1902. doi: 10.3748/wjg.v18.i16.1892
[38]
Brooks H, Lebleu B, Vives E (2005) Tat peptide-mediated cellular delivery: back to basics. Adv Drug Deliv Rev 57: 559–577. doi: 10.1016/j.addr.2004.12.001
[39]
Song YC, Sun GH, Lee TP, Huang JC, Yu CL, et al. (2008) Arginines in the CDR of anti-dsDNA autoantibodies facilitate cell internalization via electrostatic interactions. Eur J Immunol 38: 3178–3190. doi: 10.1002/eji.200838678
[40]
Srinivas S, Watanabe T, Lin CS, William CM, Tanabe Y, et al. (2001) Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev Biol 1: 4.
[41]
Schlabach MR, Hu JK, Li M, Elledge SJ (2010) Synthetic design of strong promoters. Proc Natl Acad Sci U S A 107: 2538–2543. doi: 10.1073/pnas.0914803107
[42]
Postic C, Shiota M, Niswender KD, Jetton TL, Chen Y, et al. (1999) Dual roles for glucokinase in glucose homeostasis as determined by liver and pancreatic beta cell-specific gene knock-outs using Cre recombinase. J Biol Chem 274: 305–315. doi: 10.1074/jbc.274.1.305
