Rescue of the albino phenotype by introducing a functional tyrosinase minigene into Kunming albino mice.

AIM: To use the tyrosinase minigene as a visual marker to perform microinjection training and improve the techniques related with transgene to greatly elevate the efficiency of gene transfer. METHODS: A mouse tyrosinase minigene, i.e., TyBS, in which the 2.25-kb authentic genomic 5' non-coding flanking sequence of mouse tyrosinase was fused to a mouse tyrosinase cDNA, was introduced into the fertilized eggs of outbred Kunming albino mice. RESULTS: Of the 11 animals that developed from the injected eggs, two mice (P1 and #8) exhibited pigmented hair (P1) and eyes (P1 and #8), as confirmed by PCR analysis for the tyrosinase minigene integrated into the genome. When founder P1 was bred to Kunming male mouse, six progeny out of 11 offspring inherited the transgene and the pigmented-eye phenotype. CONCLUSION: Taken together, these results suggest that this minigene encodes the active tyrosinase protein and that its 5' flanking region contains the sequences regulating the expression of mouse tyrosinase gene as expected. We have rescued the albino phenotype by introduction and expression of a functional tyrosinase minigene in the Kunming albino mouse and the transgene can be passed to subsequent generation. These findings also indicate that TyBS can be a useful visual marker gene in the co-transgenic experiments.

Generation of the regulatory protein rtTA transgenic mice.

AIM: To translate Tet-on system into a conditional mouse model, in which hepatitis B or C virus (HBV or HCV) gene could be spatiotemporally expressed to overcome "immune tolerance" formed during the embryonic development and "immune escape" against hepatitis virus antigen(s), an effector mouse, carrying the reverse tetracycline-responsive transcriptional activator (rtTA) gene under the tight control of liver-specific human apoE promoter, is required to be generated. METHODS: To address this end, rtTA fragment amplified by PCR was effectively inserted into the vector of pLiv.7 containing apoE promoter to create the rtTA expressing vector, i.e., pApoE-rtTA. ApoE-rtTA transgenic fragment (-6.9 kb) released from pApoE-rtTA was transferred into mice by pronucleus injection, followed by obtaining one transgene (+) founder animal from microinjection through PCR and Southern blot analysis. RESULTS: rtTA transgene which could be transmitted to subsequent generation (F1) derived from founder was expressed in a liver-specific fashion. CONCLUSION: Taken together, these findings demonstrate that rtTA transgenic mice, in which rtTA expression is appropriately targeted to the murine liver, are successfully produced, which lays a solid foundation to 'off-on-off' regulate expression of target gene (s) (e.g., HBV and/or HCV) in transgenic mice mediated by Tet-on system.

[siRNAs targetting sphingomyelin phosphodiesterase 1 protect mouse oocytes from apoptosis].

OBJECTIVE: To investigate the potential of siRNAs targeting sphingomyelin phosphodiesterase 1 (SMPD1) in protecting the oocytes from apoptosis, and explore new approaches to female fertility preservation. METHODS: Chemically synthesized siRNA targeting SMPD1 were introduced into mouse oocytes retrieved by hyperstimulation, and the cell apoptosis was analyzed by comic assay 48 and 72 h later. RESULTS: In the oocytes without any siRNA injection, oocyte DNA damage occurred after 24 h, and large amount of DNA fragments migrated from the cells 48 h later. In oocytes injected with siRNA003, DNA migration decreased significantly as compared with the control and the other two groups injected with siRNA001 and siRNA002 (P<0.01). CONCLUSION: siRNA targeting SMPD1 may protect the oocytes from apoptosis, and has the potential for use in future female fertility preservation.

Temporal control of Cre recombinase-mediated in vitro DNA recombination by Tet-on gene expression system.

Conditional gene expression and gene deletion are important experimental approaches for examining the functions of particular gene products in mouse models. These strategies exploiting Cre-mediated site-specific DNA recombination have been incorporated into transgenic and gene-targeting procedures to allow in vivo manipulation of DNA in embryonic stem cells (ES cells) or living animals. The Cre/lox P system has become widely used in conditional gene targeting, conditional gene repair and activation, inducible chromosome translocation, and chromosome engineering. In this project, we have employed the universal transgenic system and the liver-specific promoter system for tightly temporal and liver-specific control of Cre gene expression in mice that (1) integrates the advantages of the Tet-on gene expression system and Cre/lox P site-mediated gene activation, and (2) simplifies the scheme of animal crosses through a combination of two control elements in a single transgene. A liver-specific apoE promoter was inserted into the promoter cloning site upstream of the rtTA cassette of pCore construct to generate the transgene construct pApoErtTA-tetO-Cre, followed by demonstrating stringent regulation of doxycycline (Dox)-induced Cre-mediated recombination in the lox P-flanked transcription STOP cassette-modified BEL-7402 cells. That is to say, in the absence of Dox, the Cre gene is not expressed and will not induce site-specific recombination between two lox P sites, whereas on exposure to Dox, the Cre gene will be expressed and the recombination will occur. Together, these data indicate that the Tet-on gene expression system is able to successfully and stringently control Cre expression in vitro, which lays a solid foundation for efficient and spatio-temporal Cre gene activation in transgenic mice.

Temporal and tight hepatitis C virus gene activation in cultured human hepatoma cells mediated by a cell-permeable Cre recombinase.

Conditional gene expression has greatly facilitated the examination of the functions of particular gene products. Using the Cre/lox P switching expression system, we plan to develop efficient conditional transgene activation of hepatitis C virus core protein (HCV-C) cDNA (nucleotide 342-914) in the transgenic mice to overcome "immune tolerance" formed during the embryonic period and "immune escape" against hepatitis virus antigen in our project. To use this system in vivo, the dormant transgenic construct, i.e., pApoE-SCS-EGFP-HCV-C, was generated using techniques of standard molecular biology. The liverspecific human apoE promoter was here used to target expression of genes of interest (EGFP and HCV-C) to murine liver. Prior to generating the transgenic mice, the availability of Cre/lox P system and construct functionality were successfully verified by a cell-free recombination system and via checking the expression of EGFP and HCV-C in the human hepatoma cells at the mRNA and protein levels. These results suggest that the Cre/lox P system could tightly control expression of EGFP and HCV-C in vitro, which laid a solid foundation to conditionally activate expression of target gene(s) in transgenic mice by Cre-mediated site-specific recombination.