Therapeutic Effects of Hematopoietic Stem Cell Derived From Gene-Edited Mice on ß654-Thalassemia.

ß-thalassemia is an inherited blood disease caused by reduced or inadequate ß-globin synthesis due to ß-globin gene mutation. Our previous study developed a gene-edited mice model (ß654-ER mice) by CRISPR/Cas9-mediated genome editing, targeting both the ßIVS2-654 (C > T) mutation site and the 3' splicing acceptor site at 579 and corrected abnormal ß-globin mRNA splicing in the ß654-thalassemia mice. Herein, we further explored the therapeutic effect of the hematopoietic stem cells (HSCs) from ß654-ER mice on ß-thalassemia by consecutive HSC transplantation. The results indicated that HSC transplantation derived from gene-edited mice can significantly improve the survival rate of mice after lethal radiation doses and effectively achieve hematopoietic reconstruction and long-term hematopoiesis. Clinical symptoms, including hematologic parameters and tissue pathology of transplanted recipients, were significantly improved compared to the non-transplanted ß654 mice. The therapeutic effect of gene-edited HSC transplantation demonstrated no significant difference in hematological parameters and tissue pathology compared with wild-type mouse-derived HSCs. Our data revealed that HSC transplantation from gene-edited mice completely recovered the ß-thalassemia phenotype. Our study systematically investigated the therapeutic effect of HSCs derived from ß654-ER mice on ß-thalassemia and further confirmed the efficacy of our gene-editing approach. Altogether, it provided a reference and primary experimental data for the clinical usage of such gene-edited HSCs in the future.

Missense mutation of c.635 T > C in CAPN3 impairs muscle injury repair in a Limb-Girdel Muscular Dystropy Model.

Limb-girdle muscular dystrophy recessive 1 (LGMDR1), previously known as LGMD2A, is a specific LGMD caused by a gene mutation encoding the calcium-dependent neutral cysteine protease calpain-3 (CAPN3). In our study, the compound heterozygosity with two missense variants c.635 T > C (p.Leu212Pro) and c.2120A > G (p.Asp707Gly) was identified in patients with LGMDR1. However, the pathogenicity of c.635 T > C has not been investigated. To evaluate the effects of this novel likely pathogenic variant to the motor system, the mouse model with c.635 T > C variant was prepared by CRISPR/Cas9 gene editing technique. The pathological results revealed that a limited number of inflammatory cells infiltrated the endomyocytes of certain c.635 T > C homozygous mice at 10 months of age. Compared with wild-type mice, motor function was not significantly impaired in Capn3 c. 635 T > C homozygous mice. Western blot and immunofluorescence assays further indicated that the expression levels of the Capn3 protein in muscle tissues of homozygous mice were similar to those of wild-type mice. However, the arrangement and ultrastructural alterations of the mitochondria in the muscular tissues of homozygous mice were confirmed by electron microscopy. Subsequently, muscle regeneration of LGMDR1 was simulated using cardiotoxin (CTX) to induce muscle necrosis and regeneration to trigger the injury modification process. The repair of the homozygous mice was significantly worse than that of the control mice at day 15 and day 21 following treatment, the c.635 T > C variant of Capn3 exhibited a significant effect on muscle regeneration of homozygous mice and induced mitochondrial damage. RNA-sequencing results demonstrated that the expression levels of the mitochondrial-related functional genes were significantly downregulated in the mutant mice. Taken together, the results of the present study strongly suggested that the LGMDR1 mouse model with a novel c.635 T > C variant in the Capn3 gene was significantly dysfunctional in muscle injury repair via impairment of the mitochondrial function.

Maternal Prkce expression in mature oocytes is critical for the first cleavage facilitating maternal-to-zygotic transition in mouse early embryos.

OBJECTIVES: Early embryo development is dependent on the regulation of maternal messages stored in the oocytes during the maternal-to-zygote transition. Previous studies reported variability of oocyte competence among different inbred mouse strains. The present study aimed to identify the maternal transcripts responsible for early embryonic development by comparing transcriptomes from oocytes of high- or low- competence mouse strains. MATERIALS AND METHODS: In vitro fertilization embryos from oocytes of different mouse strains were subject to analysis using microarrays, RNA sequencing, real-time quantitative PCR (RT-qPCR) analysis, Western blotting, and immunofluorescence. One candidate gene, Prkce, was analysed using Prkce knockout mice, followed by a cRNA rescue experiment. RESULTS: The fertilization and 2-cell rate were significantly higher for FVB/NJ (85.1% and 82.0%) and DBA/2J (79.6% and 76.7%) inbred mouse strains than those for the MRL/lpr (39.9% and 35.8%) and 129S3 (35.9% and 36.6%) strains. Thirty-nine differentially expressed genes (DEGs) were noted, of which nine were further verified by RT-qPCR. Prkce knockout mice showed a reduced 2-cell rate (Prkce+/+ 80.1% vs. Prkce-/- 32.4%) that could be rescued by Prkce cRNA injection (2-cell rate reached 76.7%). Global transcriptional analysis revealed 143 DEGs in the knockout mice, which were largely composed of genes functioning in cell cycle regulation. CONCLUSIONS: The transcription level of maternal messages such as Prkce in mature oocytes is associated with different 2-cell rates in select inbred mouse strains. Prkce transcript levels could serve as a potential biomarker to characterize high-quality mature oocytes.

Correction of RNA splicing defect in ß654-thalassemia mice using CRISPR/Cas9 gene-editing technology.

ß654-thalassemia is a prominent Chinese subtype of b-thalassemia, representing 17% of all cases of ß-thalassemia in China. The molecular mechanism underlying this subtype involves the IVS-2-654 C→T mutation leading to aberrant ß-globin RNA splicing. This results in an additional 73-nucleotide exon between exons 2 and 3 and leads to a severe thalassemia syndrome. Herein, we explored a CRISPR/Cas9 genome editing approach to eliminate the additional 73- nucleotide by targeting both the IVS-2-654 C→T and a cryptic acceptor splice site at IVS-2-579 in order to correct aberrant b-globin RNA splicing and ameliorate the clinical ß-thalassemia syndrome in ß654 mice. Gene-edited mice were generated by microinjection of sgRNA and Cas9 mRNA into one-cell embryos of ß654 or control mice: 83.3% of live-born mice were gene-edited, 70% of which produced correctly spliced RNA. No off-target events were observed. The clinical symptoms, including hematologic parameters and tissue pathology of all of the edited ß654 founders and their offspring were significantly improved compared to those of the non-edited ß654 mice, consistent with the restoration of wild-type b-globin RNA expression. Notably, the survival rate of gene-edited heterozygous ß654 mice increased significantly, and liveborn homozygous ß654 mice were observed. Our study demonstrated a new and effective gene-editing approach that may provide groundwork for the exploration of ß654-thalassemia therapy in the future.

ß-Galactosidase gene codon optimization results in post-transcriptional enhancement of expression.

OBJECTIVE: lacZ encodes for ß-galactosidase within the galactose operon of bacterial cells. When used as a reporter gene, bacterial "ß-galactosidase" expression is often insufficient for detection in mammalian cells. We intended to optimize the lacZ codon usage according to the most frequently used codons for the seven major proteins in cow's milk, in order to pave a way for the enhancement of transgenic genes expression in eukaryotes. RESULTS: We constructed modified lacZ (named olacZ) according to optional codons used for proteins expressed in cow's milk. The expression of lacZ and olacZ was then compared in HC11 (a murine mammary gland epithelial line), 293T, HeLa, Cos7, and NIH 3T3 cells. While there was no significant difference at the mRNA level between lacZ and olacZ (P > 0.05). The quantification of ß-galactosidase activity and in situ staining experiments showed a 1.2-fold to 3.3-fold expression improvement when comparing olacZ with lacZ. The levels of ß-galactosidase expression at the protein levels from olacZ were approximately 9.2-fold and 2.4-fold respectively for Cos7 and HC11 cells. Furthermore, a 1.9-fold tendency of enhanced expression of olacZ in mammary gland during lactation was observed in transgenic-olacZ mice. CONCLUSION: This study demonstrates an alternative choice for improving lacZ reporter expression in eukaryotes, especially in the mammary gland of cattle or goats.

Production of biologically active human factor IX-Fc fusion protein in the milk of transgenic mice.

OBJECTIVE: To investigate the feasibility of producing human IgG1 Fc fragment fused factor IX (FIX-Fc) in the milk of transgenic animals, for an alternative possible solution to the unmet need of FIX-Fc products for hemophilia B treatment. RESULTS: Six founder lines of transgenic mice harboring FIX-Fc cassette designed to be expressed specifically in the mammary gland were generated. FIX-Fc protein was secreted into the milk of transgenic mice with preserved biological activity (with the highest value of 6.2 IU/mL), similar to that of the non-fused FIX transgenic milk. RT-PCR and immunofluorescence analysis confirmed that FIX-Fc was specifically expressed in the mammary gland. The blood FIX clotting activities were unchanged, and no apparent health defects were observed in the transgenic mice. Moreover, the stability of FIX protein in milk was increased by the Fc fusion. CONCLUSIONS: It is feasible to produce biologically functional FIX-Fc in the mammary gland of transgenic mice. Our preliminary results provide a foundation for the potential scale-up production of FIX-Fc in the milk of dairy animals.

Efficient stabilization of recombinant human coagulation factor VIII in the milk of transgenic mice using hFVIII and vWF co-expression vector transduction.

OBJECTIVES: To investigate the reasons for the instability of human coagulation factor FVIII (hFVIII) in milk which is an intractable obstacle during the hFVIII production by a transgenic mammary gland bioreactor. RESULTS: We constructed P1A3-hFVIIIBDD and P1A3-hFVIIIBDD-IRES-vWF co-expression cassettes for generating transgenic mice. P1A3-hFVIII/CMV-vWF double heterozygotes were also prepared by mating P1A3-hFVIIIBDD with CMV-vWF mice. hFVIII bioactivity in milk was determined under different storage conditions. The half-life (in vitro) of hFVIII bioactivity in P1A3-hFVIIIBDD-IRES-vWF mice was significantly longer than P1A3-hFVIIIBDD mice [77 ± 4.9 vs. 44 ± 2.6 h at 4 °C, 32.5 ± 5 vs. 19.7 ± 0.6 h at room temperature and 7.4 ± 1.4 vs. 3.4 ± 0.6 at 37 °C, respectively (P < 0.05)]. The half-life (in vitro) of hFVIII bioactivity in milk of double heterozygotes was similar to P1A3-hFVIIIBDD-IRES-vWF ones, demonstrating that the vWF transgene expression in hFVIII transgenic mice can efficiently improve the stabilization of hFVIII bioactivity in milk. CONCLUSION: We provide a new approach of P1A3-hFVIIIBDD-IRES-vWF co-expression to generate more stable hFVIII in transgenic milk with rapid and low cost as well as valuable information for producing pharmaceutical proteins by transgenic mammary gland bioreactor.

Induction of protection against foot-and-mouth disease virus in cell culture and transgenic suckling mice by miRNA targeting integrin αv receptor.

Foot-and-mouth disease virus (FMDV) is an RNA virus that causes a highly contagious disease in domestic and wild cloven-hoofed animals. Although vaccination has been used to protect animals against FMDV, there are shortcomings in the efficacy of the available vaccines. RNA interference (RNAi) is triggered by small RNA molecules, including short interfering RNAs and microRNAs (miRNAs), and the use of RNAi-based methods have demonstrated promise as an alternative method of controlling the transmission of FMDV. However, the method of delivery, short duration of siRNA and miRNA in vivo, and the genetic variability of FMDV confound the use of RNAi-based strategies for FMDV control. FMDV has been shown to exploit host-cell integrins as cell-surface receptors to initiate infection. We selected the gene for the integrin αv subunit as an RNAi target, and constructed three αv-specific miRNA expression plasmids. The effects of these miRNAs on FMDV infection were examined in PK-15 cells and transgenic suckling mice. In PK-15 cells, the expression of the αv-specific miRNAs significantly inhibited the expression of integrin αv receptor and decreased FMDV infection. The transgenic mice were generated by integrating the αv-specific miRNA expression cassette using pronuclear microinjection. When challenged with a dose of FMDV ten times greater than the LD50, the survival rate of transgenic suckling mice was approximately six-fold higher than that of their non-transgenic littermates, indicating that the interference of the miRNAs significantly reduced FMDV infection in the transgenic mice. This is the first report of limiting FMDV attachment to cellular receptors using miRNA-mediated gene knock down of cell-surface receptors to significantly reduce FMDV infection in cell culture and transgenic suckling mice.

Therapeutic effects of induced pluripotent stem cells in chimeric mice with ß-thalassemia.

Although ß-thalassemia is one of the most common human genetic diseases, there is still no effective treatment other than bone marrow transplantation. Induced pluripotent stem cells have been considered good candidates for the future repair or replacement of malfunctioning organs. As a basis for developing transgenic induced pluripotent stem cell therapies for thalassemia, ß(654) induced pluripotent stem cells from a ß(654) -thalassemia mouse transduced with the normal human ß-globin gene, and the induced pluripotent stem cells with an erythroid-expressing reporter GFP were used to produce chimeric mice. Using these chimera models, we investigated changes in various pathological indices including hematologic parameters and tissue pathology. Our data showed that when the chimerism of ß(654) induced pluripotent stem cells with the normal human ß-globin gene in ß(654) mice is over 30%, the pathology of anemia appeared to be reversed, while chimerism ranging from 8% to 16% provided little improvement in the typical ß-thalassemia phenotype. Effective alleviation of thalassemia-related phenotypes was observed when chimerism with the induced pluripotent stem cells owning the erythroid-expressing reporter GFP in ß(654) mouse was greater than 10%. Thus, 10% or more expression of the exogenous normal ß-globin gene reduces the degree of anemia in our ß-thalassemia mouse model, whereas treatment with ß(654) induced pluripotent stem cells which had the normal human ß-globin gene had stable therapeutic effects but in a more dose-dependent manner.

Expression of human transferrin can be regulated effectively by rabbit transferrin regulatory elements in transgenic mice.

Human transferrin (hTF) belongs to the iron-binding glycoprotein family. It plays an important role in iron transport throughout the body. Transgenic mice are a good model to study how to produce functional hTF on a large-scale. We have improved the expression of hTF and investigated its regulatory mechanism in transgenic mice. Three expression constructs were prepared in which hTF expression was controlled by different regulatory cassettes of rabbit transferrin (rTF). hTF was secreted into serum of transgenic mice when its expression was controlled by the rTF promoter and enhancer, whereas the rTF enhancer in tandem with the rTF promoter repressed hTF secretion into milk. A significant inverse relationship between methylation of the rTF promoter and hTF expression was observed in liver, heart, mammary gland, and muscle of transgenic mice. The highest concentration of hTF was 700 µg/ml in milk.

Transgenically mediated shRNAs targeting conserved regions of foot-and-mouth disease virus provide heritable resistance in porcine cell lines and suckling mice.

Foot-and-mouth disease virus (FMDV) is responsible for substantial economic losses in livestock breeding each year, and the development of new strategies is needed to overcome the limitations of existing vaccines and antiviral drugs. In this study, we evaluated the antiviral potential of transgenic porcine cells and suckling mice that simultaneously expressed two short-hairpin RNAs (shRNAs) targeting the conserved regions of the viral polymerase protein 3D and the non-structural protein 2B. First, two recombinant shRNA-expressing plasmids, PB-EN3D2B and PB-N3D2B, were constructed and the efficiency of the constructs for suppressing an artificial target was demonstrated in BHK-21 cells. We then integrated PB-EN3D2B into the genome of the porcine cell line IBRS-2 using the piggyBac transposon system, and stable monoclonal transgenic cell lines (MTCL) were selected. Of the 6 MTCL that were used in the antiviral assay, 3 exhibited significant resistance with suppressing ratios of more than 94% at 48 hours post-challenge (hpc) to both serotype O and serotype Asia 1 FMDV. MTCL IB-3D2B-6 displayed the strongest antiviral activity, which resulted in 100% inhibition of FMDV replication until 72 hpc. Moreover, the shRNA-expressing fragment of PB-N3D2B was integrated into the mouse genome by DNA microinjection to produce transgenic mice. When challenged with serotype O FMDV, the offspring of the transgenic mouse lines N3D2B-18 and N3D2B-81 exhibited higher survival rates of 19% to 27% relative to their non-transgenic littermates. The results suggest that these heritable shRNAs were able to suppress FMDV replication in the transgenic cell lines and suckling mice.

Directed neuronal differentiation of mouse embryonic and induced pluripotent stem cells and their gene expression profiles.

Embryonic stem cells (ESCs) may be useful as a therapeutic source of cells for the production of healthy tissue; however, they are associated with certain challenges including immunorejection as well as ethical issues. Induced pluripotent stem cells (iPSCs) are a promising substitute since a patient's own adult cells would serve as tissue precursors. Ethical concerns prevent a full evaluation of the developmental potency of human ESCs and iPSCs, therefore, mouse iPSC models are required for protocol development and safety assessments. We used a modified culturing protocol to differentiate pluripotent cells from a mouse iPS cell line and two mouse ES cell lines into neurons. Our results indicated that all three pluripotent stem cell lines underwent nearly the same differentiation process when induced to form neurons in vitro. Genomic expression microarray profiling and single-cell RT-qPCR were used to analyze the neural lineage differentiation process, and more than one thousand differentially expressed genes involved in multiple molecular processes relevant to neural development were identified.

Bovine prolactin promotes the expression of human transferrin in the milk of transgenic mice.

The bovine prolactin vector was injected directly into the mammary glands of mice carrying the human transferrin transgene to investigate its effect on the production of human transferrin in milk. The mean levels of human transferrin in two experimental groups were increased by approx. 60% compared with the control group: 1143 +/- 196 ng/ml (experimental group 1; two injections) and 1160 +/- 189 ng/ml (experimental group 2; three injections) versus 714 +/- 75 ng/ml (control group). These findings suggest the potential utility of the prolactin vector for efficient expression of valuable pharmaceutical proteins in transgenic animal mammary glands.

Transgenic human alpha-hemoglobin stabilizing protein could partially relieve betaIVS-2-654-thalassemia syndrome in model mice.

Beta-thalassemia is an anemia caused by a relative excess of alpha-hemoglobin (alphaHb) due to absent or reduced beta-hemoglobin (betaHb) synthesis. In this study, we explore whether the introduction of alpha-hemoglobin stabilizing protein (AHSP), a chaperone protein for proper folding and stabilization of free alphaHb in red blood cells, thus aiding hemoglobin A (HbA) assembly, could relieve the pathogenic state of red blood cells in beta-thalassemia. For that, a human ahsp vector was constructed to generate transgenic human ahsp mice in a model of beta(IVS-2-654)-thalassemia by microinjecting the vector into fertilized eggs, resulting in the production of double heterozygous mice (h-ahsp(+)/beta(IVS-2-654+)). Real-time quantitative RT-PCR and Western blot analysis confirmed AHSP expression in three h-ahsp(+)/beta(IVS-2-654+) mice. Hematologic determination showed an improvement in the red blood cell indices of these h-ahsp(+)/beta(IVS-2-654+) mice. The red blood cell count and hemoglobin level were elevated to various extents as compared with their diseased siblings. A dramatic reduction in anisocytosis in the peripheral blood of h-ahsp(+)/beta(IVS-2-654+) mice was observed (16.2 +/- 4.6 vs. 30.0 +/- 5.2%). Few erythroid precursors appeared in the liver sinusoids of h-ahsp(+)/beta(IVS-2-654+) mice. Splenomegaly with extramedullary hematopoiesis was also ameliorated. Significantly, serum iron concentration was remarkably reduced as compared with that of h-ahsp(-)/beta(IVS-2-654+) mice (43.2 +/- 14.9 vs. 82.4 +/- 12.9 microM), and iron deposition in the liver was decreased in h-ahsp(+)/beta(IVS-2-654+) mice. All these results suggested amelioration of the anemia phenotype in h-ahsp(+)/beta(IVS-2-654+) mice after introduction of the ahsp gene. We therefore propose that an ahsp transgene could provide an adjuvant method for gene therapy of beta-thalassemia.

[Construction of the oocyte-specific expressing phiC31 integrase vector pZP3-INT and its expression in mouse oocytes].

Streptomyces phage phiC31 integrase is a site-specific recombinase, which can catalyze site-specific, unidirectional recombination between the attP site and attB site. To explore whether it can be used to mediate the recombination of specific gene in oocytes, GV-stage oocytes were collected from 3-week-old Kunming White mice by puncturing antral follocles with a sharp needle, and micro-injected with oocyte-specific expressing phiC31 integrase vector pZP3-INT and site -specific recombination detection vector pBCPB+. phiC31 integrase mRNA were detected by RT-PCR and the recombination of pBCPB+ was evaluated by PCR in mouse oocytes at 48 h after injection. Both can get corresponding bands. These results indicated that the expression of phiC31 integrase can be driven by ZP3 promoter efficiently and phiC31 integrase can mediate the site-specific recombination between attP site and attB site in mouse GV-stage oocytes. It could be a powerful tool for the study of recombination of specific gene in mouse oocytes and would provide an alternative way for the mouse oocyte genome manipulation.

Association of differential and site-dependent CpG methylation and diverse expression of DNA methyltransferases with the tissue-specific expression of human beta-globin gene in transgenic mice.

Expression of human locus control region (LCR) and beta-globin promoter has been recognized as an important factor in time- and tissue-specific expression event. DNA methylation can affect the transcriptional activity of specific genes. To investigate the methylation mechanism in the regulation of LCR and promote expression, this study used a transgenic mouse strain generated previously, in which the hematopoietic-specific expression of the EGFP was driven by human beta-globin promoter and under the control of LCR, to examine the CpG methylation pattern in various tissues. The results showed the inverse correlation between the methylated extent and the levels of gene expression in all tested tissues. We also found that the methylated extent of the 10 examined CpG sites was biased along their positions and is more efficient near the transcription start site. Real-time quantitative RT-PCR analysis of DNA methyltransferases (DNMTs) transcripts showed that Dnmt3a and Dnmt3b expressed with a very low level in the hematopoietic tissues that was coincident with the relative higher EGFP expression in these tissues, indicating that the differential expression of DNMTs contributed to the tissue-specific methylated patterns which caused the diverse gene expression in various tissues. These findings provide significant clues to elucidate the mechanism of the regulation on tissue-specific expression of genes.

A novel transgenic mouse model produced from lentiviral germline integration for the study of beta-thalassemia gene therapy.

BACKGROUND: beta-thalassemia is one of the most common genetic diseases in the world and requires extensive therapy. Lentiviral-mediated gene therapy has been successfully exploited in the treatment of beta-thalassemia and showed promise in clinical application. Using a human beta-globin transgenic mouse line in a beta-thalassemia diseased model generated with a lentiviral-mediated approach, we investigate the stable therapeutic effect on a common thalassemia syndrome. DESIGN AND METHODS: Human beta-globin gene lentiviral vector was constr ucted, followed by subzonal microinjection into single-cell embryos of beta(IVS-2-654)-thalassemia mice to generate a transgenic line. Human beta-globin gene expression was examined with RT-PCR, Western-blotting and ELISA. The hematologic parameters and tissue pathology were investigated over time in founder mice and their off-spring. RESULTS: Transgenic mice with stable expression of the lentivirus carrying human beta-globin gene were obtained. A marked improvement in red blood cell indices and a dramatic reduction in red blood cell anisocytosis, poikilocytosis and target cells were observed. Nucleated cell proportion was greatly decreased in bone marrow, and splenomegaly with extramedullary hematopoiesis was ameliorated. Iron deposition in liver was also reduced. There was a two-fold increase in the survival rate of the beta(IVS-2-654) mice carrying human beta-globin transgene. Significantly, the germline integration of the lentiviral construct was obtained and stable hematologic phenotype correction was observed over the next two generations of the transgenic mice. CONCLUSIONS: The generation of human beta-globin transgenic mice in a beta(IVS-2-654)-thalassemia mouse mediated with lentiviral vectors provides a useful model and offers an attractive means to investigate the transgenic stable therapeutic effect in beta-thalassemia.

[Chromosomal localization of foreign genes in transgenic mice using dual-color fluorescence in situ hybridization].

OBJECTIVE: To establish a highly sensitive and specific dual-color fluorescence in situ hybridization (D-FISH) method used for chromosomal localization of foreign genes in double transgenic mice. METHODS: Two strains of double transgenic mice were used in this experiment, one was integrated with the herpes simplex virus thymidine kinase (HSV-tk) and the enhanced green fluorescence protein (eGFP), the other was with the short hairpin RNA interference(RNAi) and beta(654). Splenic cells cultured in vitro were arrested in metaphase by colchicine and hybridized with digoxigenin-labeled and biotinylated DNA probes, then detected by rhodamine-conjugated avidin and FITC-conjugated anti-digoxigenin. RESULTS: Dual-color fluorescence signals were detected on the same metaphase in both transgenic mice strains. In HSV-tk/eGFP double transgenic mice, strong green fluorescence for HSV-tk and red for eGFP were observed and localized at 2E5-G3 and 8A2-A4 respectively. In beta(654)/RNAi mice, beta(654) was detected as red fluorescence on chromosome 7D3-E2, and RNAi showed random integration on chromosomes. It was detected as green fluorescence on chromosome 12B1 in one mouse, while on 1E2.3-1F and 3A3 in the other. CONCLUSION: Highly sensitive and specific D-FISH method was established using the self-prepared DNA probes, and chromosomal localization of the foreign genes was also performed in combination with G-banding in double transgenic mice. This technology will facilitate the researches in transgenic animals and gene therapy models.