The Cre-loxP recombination system is the most widely used technology for in vivo tracing of stem or progenitor cell lineages. The precision of this genetic system largely depends on the specificity of Cre recombinase expression in targeted stem or progenitor cells. However, Cre expression in nontargeted cell types can complicate the interpretation of lineage-tracing studies and has caused controversy in many previous studies. Here we describe a new genetic lineage tracing system that incorporates the Dre-rox recombination system to enhance the precision of conventional Cre-loxP-mediated lineage tracing. The Dre-rox system permits rigorous control of Cre-loxP recombination in lineage tracing, effectively circumventing potential uncertainty of the cell-type specificity of Cre expression. Using this new system we investigated two topics of recent debates-the contribution of c-Kit cardiac stem cells to cardiomyocytes in the heart and the contribution of Sox9 hepatic progenitor cells to hepatocytes in the liver. By overcoming the technical hurdle of nonspecific Cre-loxP-mediated recombination, this new technology provides more precise analysis of cell lineage and fate decisions and facilitates the in vivo study of stem and progenitor cell plasticity in disease and regeneration.

As a natural anti-cancer alkaloid extracted from Amaryllidaceae, lycorine shows various biological effects on tumor cells. The survival rate of HL-60 cells exposed to lycorine was decreased in a dose-dependent manner with 1 lM as the 50% inhibitory concentration (IC50), cell growth was slowed down by arresting cell cycle at G2/M phase, and cell regeneration potential was inhibited. HL-60 cells exhibited typical apoptotic morphological changes, apoptotic DNA ''ladder'' pattern, and sub-G1 peak in cell phase distribution, showing apoptosis of HL-60 cells. To further understand the apoptotic molecular mechanism of lycorine on HL-60 cells, caspase activity was tested by colorimetric assay, and the expression of Bcl-2 and Bax proteins was examined by Western blotting. The increase of caspase-8, -9, -3 activities demonstrated that caspase was a key mediator of apoptotic pathways induced by lycorine. Under-expression of Bcl-2 and increase of Bax:Bcl-2 ratio showed that Bcl-2 family proteins were involved in apoptosis. Our finding suggests that lycorine can suppress leukemia growth and reduce cell survival via arresting cell cycle and inducing apoptosis of tumor cells.

The genes encoding β-actin (ACTB in human or Actb in mouse) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH in human or Gapdh in mouse) are the two most commonly used references for sample normalization in determination of the mRNA level of interested genes by reverse transcription (RT) and ensuing polymerase chain reactions (PCR). In this study, bioinformatic analyses revealed that the ACTB, Actb, GAPDH and Gapdh had 64, 69, 67 and 197 pseudogenes (PGs), respectively, in the corresponding genome. Most of these PGs are intronless and similar in size to the authentic mRNA. Alignment of several PGs of these genes with the corresponding mRNA reveals that they are highly homologous. In contrast, the hypoxanthine phosphoribosyltransferase-1 gene (HPRT1 in human or Hprt in mouse) only had 3 or 1 PG, respectively, and the mRNA has unique regions for primer design. PCR with cDNA or genomic DNA (gDNA) as templates revealed that our HPRT1, Hprt and GAPDH primers were specific, whereas our ACTB and Actb primers were not specific enough both vertically (within the cDNA) and horizontally (compared cDNA with gDNA). No primers could be designed for the Gapdh that would not mis-prime PGs. Since most of the genome is transcribed, we suggest to peers to forgo ACTB (Actb) and GAPDH (Dapdh) as references in RT-PCR and, if there is no surrogate, to use our primers with extra caution. We also propose a standard operation procedure in which design of primers for RT-PCR starts from avoiding mis-priming PGs and all primers need be tested for specificity with both cDNA and gDNA.