A white-light-emitting sensor array based on cucurbit[8]uril for quantitative detection of multicomponent nitroaniline isomers.

A white-light-emitting supramolecular complex through supramolecular interactions has been assembled; the white luminescent supramolecular complex exhibits two emission spectra. Based on this, a dual-channel white-light array sensor was constructed. The results show that it can quickly identify and detect nitroaniline isomer pollutants (p-nitroaniline, m-nitroaniline, o-nitroaniline). When these three nitroaniline isomers were added to the supramolecular white-light array sensor, the fluorescence intensity of the white-light complex decreased to varying degrees. Linear discriminant analysis (LDA) showed that the supramolecular white-light array sensor could recognize and distinguish three nitroaniline isomers and could classify mixtures containing different concentrations. Factor 1 of the array had a good linear relationship with the concentration of pollutants, and the detection limit (LOD) was as low as 0.7 µM. The method has good reproducibility and stability. In addition, it can also qualitatively detect the nitroaniline isomers in river water and contaminated rice seedling extract. It provides an ideal platform for constructing multiresponse sensors.

Engraftment of genetically modified human amniotic fluid-derived progenitor cells to produce coagulation factor IX after in utero transplantation in mice.

Human amniotic fluid derived progenitor cells (hAFPCs) may be multipotent and can be considered a potential tool in the field of cell therapy for haemophilia B. Their capacity to express human coagulation factor IX (hFIX) after transduction and their fate after in utero transplantation is unknown. hAFPCs isolated from second trimester pregnancies were assessed for their phenotypic markers, multilineage capacity, and expression of hFIX after transduction. Their engraftment potential was analysed in a mouse model after in utero transplantation at embryonic day 12.5. Immunohistochemistry, fluorescence in situ, ELISA and PCR were used to assess post-transplant chimeras. hAFPCs expressed several pluripotent markers, including NANOG, SOX2, SSEA4 and TRA-1-60, and could differentiate into adipocytes and osteocytes. In vitro, after transduction with hFIX and EGFP cDNAs, constitutive hFIX protein expression and clotting activity were found. Engraftment was achieved in various foetal tissues after in utero transplantation. Safe engraftment without oncogenesis was confirmed, with low donor cell levels, but persistent engraftment, into different organs (liver, heart and lung) through to 12 weeks of age. Transgenic expression of circulating hFIX was detected in recipient mice for up to 12 weeks. hAFPCs can be engrafted long-term in immunocompetent mice after in utero transplantation. Thus, cell transplantation approaches using genetically engineered hAFPCs may prove valuable for the prenatal treatment for haemophilia B.

Supramolecular Fluorescence Sensor Array Based on Cucurbit[8]uril Complexes Used for the Detection of Multiplex Quaternary Ammonium Pesticides.

The simultaneous detection of multiple quaternary ammonium pesticides (QAPs) in water is a challenge due to their high solubility in water and similar structures. In this paper, we have developed a quadruple-channel supramolecular fluorescence sensor array for the simultaneous analysis of five QAPs, including paraquat (PQ), diquat (DQ), difenzoquat (DFQ), mepiquat (MQ), and chlormequat (CQ). Not only were QAP samples of different concentrations (10, 50, and 300 µM) in water distinguished with 100% accuracy but also single QAP and binary QAP mixed samples (DFQ-DQ) were sensitively quantified. Our experimental interference study confirmed that the developed array has good anti-interference ability. The array can quickly identify five QAPs in river and tap water samples. In addition, it also qualitatively detected QAP residues in Chinese cabbage and wheat seedlings extract. This array has rich output signals, low cost, easy preparation, and simple technology, demonstrating great potential in environmental analysis.

Supramolecular fluorescent probe based on a host-guest complex for amino acids recognition and detection in aqueous solution.

A supramolecular fluorescent probe based on a host-guest complex has been developed for amino acid recognition and detection in aqueous solution. Cucurbit[7]uril (Q[7]) with 4-(4-dimethylamino-styrene) quinoline (DSQ) formed a fluorescent probe (DSQ@Q[7]). The DSQ@Q[7] fluorescent probe nearly generated changes in fluorescence in response to four amino acids (arginine, histidine, phenylalanine and tryptophan). These changes were attributed to the host-guest interaction between DSQ@Q[7] and amino acids, which occurred as a consequence of the subtle cooperation of ionic dipole and hydrogen bonding. Linear discriminant analysis showed that the fluorescent probe could recognize and distinguish four amino acids, and a mixture with different concentration ratios could be well categorized in ultrapure water and tap water.

A supramolecular fluorescence array sensor for toxic heavy metal ion detection in environmental water and rice seedling extracts.

A supramolecular fluorescence array sensor based on cucurbituril-dye host-guest complexes (6-QAA@Q[7], PyY@Q[7], and TO@Q[8]) was constructed. The results showed that it can quickly identify and detect toxic heavy metal ions, such as Ag+, Cr3+, Hg2+, Ni2+, and Pb2+. When these five toxic heavy metal ions were added to the supramolecular fluorescence array sensor, different fluorescence responses were produced due to the different binding capacities of the metal ions to the cucurbituril-dye complex. Linear discriminant analysis (LDA) showed that the supramolecular fluorescence array sensor could identify and distinguish these five toxic heavy metal ions and a mixture containing different concentration ratios could be classified. The linear correlation between the metal ion concentration and factor 1 (F1) was strong, and the detection limit (LOD) was as low as 10-6-10-7 mol L-1. These five toxic heavy metal ions in environmental water and rice seedling extracts were identified using the supramolecular fluorescence array sensor. This sensor provides a quick and convenient method for monitoring toxic heavy metal ions in sewage.

A colorimetric supramolecular sensor array based on charge-transfer complexes for multiplex aniline and phenolic pollutants detection.

In order to effectively monitor toxic and harmful substances in sewage discharge, a rapid, highly sensitive detection of complex pollutants with similar structures has become an urgent problem to be solved. In this paper, a supramolecular colorimetric array sensor based on charge-transfer complex was constructed, which can quickly detect aniline and phenol pollutants (such as p-phenylenediamine, m-phenylenediamine, o-phenylenediamine, m-aminophenol, hydroquinone, and resorcinol) with similar structures. When six anilines and phenol isomers with similar structures were added to the supramolecular colorimetric array sensor, different color changes were produced under natural light. Linear discriminant analysis (LDA) showed that the supramolecular colorimetric array sensor could recognize and distinguish these isomers, and a mixture with different concentration ratios could be well categorized. The total Euclidean distance (TED) of an array with pollutant concentrations had a good linear correlation, and the detection limit (LOD) was as low as 10-5-10-6 mol L-1. Six anilines and phenol isomers in real samples were identified by supramolecular colorimetric array sensor. 1H NMR results showed that the formation of charge transfer complexes in Q[8] cavity may be the cause of color change. This work provides a fast and convenient experimental basis for monitoring the complex structure pollutants in sewage discharge.

Lab-on-a-Molecule Probe: Multitarget Detection of Five Aromatic Pesticides Using a Supramolecular Probe under Single Wavelength Excitation.

In order to prevent and control the effects of pesticide residues on human health and the ecological environment, the rapid, highly sensitive, and selective detection of multiple pesticide residues has become an urgent problem to be solved. Herein, a lab-on-a-molecule probe based on a host-guest complex (ThT@Q[8] probe) has been developed to simultaneously analyze multiple aromatic pesticides under single wavelength excitation, such as fuberidazole, thiabendazole, carbendazim, thidiazuron, and tricyclazole. The fluorescence titration spectra of the ThT@Q[8] probe with the five pesticides mentioned above showed that the fluorescence intensity exhibited a good linear correlation with the pesticide concentration and the limit of detection was as low as 10-7 M. Because the ThT@Q[8] probe exhibits diverse fluorescence color changes to the five pesticides studied under a 365 nm ultraviolet lamp, we fabricated a single probe used to detect multiple analytes in the RGB triple channel by extracting the RGB variations. Principal component analysis and linear discriminant analysis proved that the ThT@Q[8] probe can recognize and distinguish five pesticides and can be applied at different concentrations. In real samples, the ThT@Q[8] probe recognized and distinguished five pesticides in tap water and Huaxi River water. The 1H NMR spectra results proved that a charge-transfer complex of ThT and pesticides in the Q[8] cavity may be formed. Moreover, we selected a test strip as a carrier to detect pesticides. The results indicate it can be used to quickly and conveniently detect different pesticides due to the rapid color change. Besides, the ThT@Q[8] probe has good cell permeability and can be used to detect pesticide residues in living cells. This work has laid the foundation for the qualitative and quantitative multitarget detection of pesticide residues.

"Turn-Off" Supramolecular Fluorescence Array Sensor for Heavy Metal Ion Identification.

A "turn-off" supramolecular fluorescence array sensor based on the host-guest complexes between fluorescence dyes and cucurbit[n]urils for sensing metal ions was developed. Three fluorescent probes (RhB@Q[7], H33342@2Q[7], and BRE@Q[7]) were used as the sensing units to construct a supramolecular fluorescence array sensor. The binding ability of the metal ions and cucurbituril-dye probes varied; therefore, the probes and metal ions produced different fluorescence responses. When combined with linear discriminant analysis (LDA), the qualitative and quantitative detection of seven metal ions was achieved. In analytical samples, the supramolecular fluorescence array sensor recognized and distinguish seven metal ions. These results provided new research ideas for the rapid analysis and real-time monitoring of different heavy metal ions.

Association Between Eating Speed and Metabolic Syndrome: A Systematic Review and Meta-Analysis.

Objective: This review aimed to systematically summarize and meta-analyze the association between eating speed and metabolic syndrome (MetS). Methods: Following the Preferred Reporting Items for Systematic Reviews, and Meta Analyses (PRISMA) guidelines, four electronic databases (PubMed, Web of Science, MEDLINE, and EMBASE) were searched until March 2021 to identify eligible articles based on a series of inclusion and exclusion criteria. Heterogeneity was examined using I 2 statistics. Using random-effects models, the pooled odds ratios (ORs), and 95% CIs were calculated to evaluate the association between eating speed with MetS and its components, including central obesity, blood pressure (BP), high-density lipoprotein cholesterol (HDL), triglyceride (TG), and fasting plasma glucose (FPG). Results: Of the 8,500 original hits generated by the systematic search, 29 eligible studies with moderate-to-high quality were included, involving 465,155 subjects. The meta-analysis revealed that eating faster was significantly associated with higher risks of MetS (OR = 1.54, 95% CI: 1.27-1.86), central obesity (OR = 1.54, 95% CI: 1.37-1.73), elevated BP (OR = 1.26, 95% CI: 1.13-1.40), low HDL (OR = 1.23, 95% CI: 1.15-1.31), elevated TG (OR = 1.29, 95% CI: 1.18-1.42), and elevated FPG (OR = 1.16, 95% CI: 1.06-1.27) compared to eating slowly. Conclusions: The results of the review indicated that eating speed was significantly associated with MetS and its components. Interventions related to decreasing eating speed may be beneficial for the management of MetS. Systematic Review Registration: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42021242213, identifier: CRD42021242213.

Donor-host mitochondrial compatibility improves efficiency of bovine somatic cell nuclear transfer.

BACKGROUND: The interaction between the karyoplast and cytoplast plays an important role in the efficiency of somatic cell nuclear transfer (SCNT), but the underlying mechanism remains unclear. It is generally accepted that in nuclear transfer embryos, the reprogramming of gene expression is induced by epigenetic mechanisms and does not involve modifications of DNA sequences. In cattle, oocytes with various mitochondrial DNA (mtDNA) haplotypes usually have different ATP content and can further affect the efficiency of in vitro production of embryos. As mtDNA comes from the recipient oocyte during SCNT and is regulated by genes in the donor nucleus, it is a perfect model to investigate the interaction between donor nuclei and host oocytes in SCNT. RESULTS: We investigated whether the in vitro development of reconstructed bovine embryos produced by SCNT would be influenced by mtDNA haplotype compatibility between the oocytes and donor cells. Embryos from homotype A-A or B-B showed significantly higher developmental ability at blastocyst stages than the heterotype A-B or B-A combinations. Post-implantation development ability, pregnancy rate up to day 90 of gestation, as well as percent of term births were higher in the homotype SCNT groups than in the heterotype groups. In addition, homotype and heterotype SCNT embryos showed different methylation patterns of histone 3-lysine 9 (H3K9) genome-wide and at pluripotency-related genes (Oct-4, Sox-2, Nanog). CONCLUSION: Both histone and DNA methylation show that homotype SCNT blastocysts have a more successful epigenetic asymmetry pattern than heterotype SCNT blastocysts, which indicates more complete nuclear reprogramming. This may result from variability in their epigenetic patterns and responses to nuclear reprogramming. This suggests that the compatibility of mtDNA haplotypes between donor cells and host oocytes can significantly affect the developmental competence of reconstructed embryos in SCNT, and may include an epigenetic mechanism.

Hematopoietic stem cell engraftment by early-stage in utero transplantation in a mouse model.

A novel intrauterine transplantation (IUT) approach was developed to improve the efficiency of engraftment of hematopoietic stem cells (HSCs). HSCs with a green fluorescent protein (GFP) reporter gene were transplanted in utero on days 12.5, 13.5 and 14.5 post coitum (p.c.). The degree of chimerism of donor cells in recipient newborn mice was examined using fluorescent microscopy, polymerase chain reaction (PCR), fluorescence-activated cell sorting (FACS), and fluorescence in situ hybridization (FISH) analyses. Microscopic examination revealed the presence of green fluorescent signal in the peripheral blood of the chimeric mice. The highest survival rate (47%) as well as the highest chimerism rate (73%) were achieved by our new approach in the newborn mice that were subjected to in utero transplantation (IUT) on day 12.5 p.c. (E12.5) compared to the conventional IUT method. FACS analysis indicated that 1.55+/-1.10% of peripheral blood cells from the newborn mice were GFP-positive donor cells. FISH showed that cells containing the donor-specific GFP sequence were present in the bone marrow (BM) of the chimeric mice. Thus, the efficiency of chimera production with this new method of IUT was significantly improved over the existing IUT techniques and instruments.

Array-MLPA: comprehensive detection of deletions and duplications and its application to DMD patients.

Multiplex ligation-dependent probe amplification (MLPA) is widely used to screen genes of interest for deletions and duplications. Since MLPA is usually based on size-separation of the amplification products, the maximum number of target sequences that can be screened in parallel is usually limited to approximately 40. We report the design of a robust array-based MLPA format that uses amplification products of essentially uniform size (100-120 bp) and distinguishes between them by virtue of incorporated tag sequences. We were thus able to increase probe complexity to 124, with very uniform product yields and signals that have a low coefficient of variance. The assay designed was used to screen the largest set studied so far (249 patients) of unrelated Duchenne muscular dystrophy (DMD) cases from the Chinese population. In a blind study we correctly assigned 98% of the genotypes and detected rearrangements in 181 cases (73%); i.e., 163 deletions (65%), 13 duplications (5%), and five complex rearrangements (2%). Although this value is significantly higher for Chinese patients than previously reported, it is similar to that found for other populations. The location of the rearrangements (76% in the major deletion hotspot) is also in agreement with other findings. The 96-well flow-through microarray system used in this research provides high-throughput and speed; hybridization can be completed in 5 to 30 minutes. Since array processing and data analysis are fully automated, array-MLPA should be easy to implement in a standard diagnostic laboratory. The universal array can be used to analyze any tag-modified MLPA probe set.

Effect of individual heifer oocyte donors on cloned embryo development in vitro.

The aim of this study was to determine the effect of individual oocyte donors on cloned embryo development in vitro. Five Holstein heifers of varied genetic origins were subject to ovum pick up (OPU) once weekly. In total, 913 oocytes were recovered from 1304 follicles. A mean of 7.7+/-0.4 oocytes was recovered per session per animal. Individual mean oocyte production varied significantly in quantity but not in quality (morphological categories) among heifers. Oocytes from individual heifers were used as recipient cytoplasm for somatic cell nuclear transfer (SCNT). Cumulus cells, collected from a single Holstein cow genetically unrelated to the oocyte donor, were used as donor cells. Although the percentage of reconstructed embryos that started to cleave was nearly constant, the percentage of cleaved embryos that developed into blastocysts showed clear individual heifer variation (61%, 51%, 31%, 28% and 24%, respectively), with a mean of 38% showing blastocyst formation. In vitro fertilization (IVF) was also conducted with oocyte from the same heifers used in SCNT. A variation of blastocyst production among individual heifers was also shown in the IVF experiment, but the rank of oocyte donor based on the blastocyst rate was changed. In conclusion, individual oocyte donor may have an effect on cloned embryo development in vitro, which differed from the effect on IVF embryos.

Construction and characteristics of 3-end enriched cDNA library from individual embryos of cattle.

To analyze stage-specific gene expression profiles of pre-implantation embryos and evaluate potential viability, techniques were adapted to generate 3-end enriched cDNA libraries from individual embryos of cattle based on RT-PCR methodology. The reproducibility of constructing a cDNA library was tested by five independent PCR experiments with specific primers for the presence of several rare genes such as DNMT1 (DNA methylation transferase 1), DNMT2, DNMT3A, Oct-4/3 (octmer-binding transcription factor), IFN-iota, IGF-2r (insulin like growth factor 2 receptor), and the housekeeping genes, H2A and beta-actin. Results indicated repeatability and that a proportion of expressed genes in the cDNA library from an individual embryo was not affected by limited PCR amplification. From the cDNA library, 134 clones were randomly selected for sequencing and showed that structure related elements accounted for 33.5% of transcripts and the energy- and metabolism-related genes were also an important component being 11.9% in the cDNA library. Approximately 14% of genes in the library were functionally unknown including greater than 5% of genes that were likely novel because there was no identity in Genbank. The frequency of structure-related genes such as beta-actin and ribosomal proteins in the cDNA library corresponded to other reports and suggested that the cDNA library constructed by RT-PCR might be proportional to the mRNA populations. The cDNA libraries constructed from different stage embryos will provide a powerful tool to explore novel genes relevant to embryogenesis, determine the profiling of stage-specific gene expression, and evaluate the potential viability of embryos.

Development of an HSV-tk transgenic mouse model for study of liver damage.

The herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system that selectively depletes cells expressing HSV-tk upon treatment with GCV has provided a valuable tool for developing a new animal model expressing the desired tissue damage. In this paper, an HSV-tk vector with an albumin promoter/enhancer was constructed. Based on the favourable killing effect on Hep-G2 cells by the recombinant construct, the HSV-tk transgenic mouse strains were developed. One strain of the TK transgenic mouse (TK5) was studied intensively. Integration of the target gene was confirmed primarily by PCR. Fluorescence in situ hybridization following G-banding analysis demonstrated that the insertion site was located at 2F1-G3. The hepatocyte-specific transcription and expression of HSV-tkwas verified by reverse transcription (RT)-PCR as well as by immunohistochemical staining. When two second-generation mice (TK5-F1 and TK5-F2) were injected with GCV, the pathogenic alterations in the liver were readily identified, including the appearance of vaculation in the hepatocytes with inflammatory infiltration in the liver, and diffuse proliferation of hepatocytes. In addition, the blood test demonstrates a significant increase of serum alanine aminotransferase, aspartate aminotransferase and total bilirubin. In conclusion, the transgenic mouse model with hepatocyte-specific expressed HSV-tk developed hepatitis with administration of GCV, had morphological and clinical chemical characteristics indicative of hepatocellular disease and should be useful for the the study of inducible liver-specific diseases.

In utero transplantation of human hematopoetic stem cells into fetal goats under B-type ultrasonographic scan: an experimental model for the study of potential prenatal therapy.

OBJECTIVE: Using fetal goats as animal models, to establish the methodology of in utero transplantation of human hematopoeitic stem cell (HSC) under B-scan ultrasonographic guidance for prenatal therapy. STUDY DESIGN: Human HSC were directly injected into the peritoneal cavities of the recipient fetal goats at 45-55 days of gestation (term: 145 days) under the guidance of B-type ultrasound scan. After birth, the peripheral blood was collected for fluorescence assisted cell sorting (FACS), quantitative real-time PCR and fluorescence in situ hybridization (FISH) to detect and analyze the presence of human cells in the recipients. RESULTS: The 32 recipients were born alive except one miscarriage. To test for the presence of human-goat chimeras, cells from 13 randomly selected transplanted goats were collected. FACS analyses showed the presence of human cells in all the transplanted goats tested. The average proportion of CD34+ cells and GPA+(glycophorin A) cells in the peripheral blood were 1.34 +/- 1.10% and 2.80 +/- 2.10%, respectively. No CD34+ or GPA+ cells were found in the non-transplanted goats tested. The results of the quantitative real-time PCR in three engraftment goats were 1.2 x 10(4), 2.9 x 10(4), and 3.2 x 10(4) copies of human GPA DNA per mug of genomic DNA. FISH experiments showed that cells containing human specific alpha-satellite DNA sequence were present in the peripheral blood of the transplanted goats. CONCLUSIONS: The method described herein is safe and reliable, with low miscarriage risk and high chimerism rate. This approach may provide a promising animal model for potential prenatal treatment.

Improving in vitro development of cloned bovine embryos with hybrid (Holstein-Chinese Yellow) recipient oocytes recovered by ovum pick up.

In the present study, oocytes from F1 hybrid cattle, as well as their parental lines, were recovered by ovum pick up (OPU) and used as recipient cytoplasm for somatic cell nuclear transfer (SCNT). Four F1 hybrid (Holstein dam x Chinese Yellow sire), 10 Holstein and four Chinese Yellow cattle were subjected to OPU once weekly. There were no significant differences among breeds for number of recovered oocytes per session (overall average, 7.8+/-0.5; mean+/-S.E.M.), quality of the recovered oocytes, or oocyte maturation rate (72-73%). Matured oocytes were all used as recipient cytoplasm (without selection) and a single batch of cumulus cells collected from a Holstein cow were used as donor cells. Although reconstructed embryos initiated cleavage sooner when the recipient cytoplasm was from hybrid cattle versus the two parental breeds, the overall cleavage rate was indistinguishable among breeds. At Day 8, the blastocyst rate from the cleaved embryos (51% versus 37% and 27%), the total number of cells per blastocyst (135+/-4.1 versus 116+/-3.6 and 101+/-4.2), and the percentage of Grade-A (excellent quality) blastocysts (54% versus 42% and 29%) in the hybrid group were all higher than that of Holstein and Yellow groups. Furthermore, the proportion of blastocysts obtained at Day 7 (as a percentage of the total number of blastocysts) was greater in the hybrid group than in Holstein and Yellow groups (89% versus 71% and 63%). In conclusion, the use of F1 hybrid oocytes as recipient cytoplasm significantly improved in vitro development of cloned bovine embryos relative to oocytes derived from the parental lines.

Construction of an allogenic chimeric mouse model for the study of the behaviors of donor stem cells in vivo.

BACKGROUND: It is essential to establish an animal model for the elucidation of the biological behaviors of stem cells in vivo. We constructed a chimeric animal model by in utero transplantation for investigation of stem cell transplantation. METHODS: This chimerism was achieved by injecting the stem cells derived from the bone marrow of green fluorescence protein (GFP)-transgenic mice into fetal mice at 13.5 days of gestation. Several methods such as polymerase chain reaction (PCR), real-time PCR, fluorescence-assisted cell sorting (FACS) and fluorescence in situ hybridization (FISH) were used for the observation of donor cells. RESULTS: Under a fluorescence microscope, we observed the GFP cells of donor-origin in a recipient. PCR, FACS analysis and FISH indicated chimerism at various intervals. Real-time PCR indicated that some donor cells existed in chimera for more than 6 months. CONCLUSIONS: Allogenic stem cells may exist in recipients for a long time and this allogenic animal model provides a useful tool for studying the behavior of hematopoietic stem cells and also offers an effective model system for the study of stem cells.

[Suppression of enhanced green fluorescent protein expression in cells by RNA interference].

OBJECTIVE: To evaluate the role of RNA interference (RNAi) in silencing the enhanced green fluorescent protein (eGFP) expression in 293T and Mel cells. METHODS: Nested-PCR was used to amplify H1 promoter from human 293T cells for driving RNAi synthesis. RNAi vectors (TR1) for silencing the eGFP expression was constructed. The eGFP vector and RNAi vector (TR1) were then co-transfected into the 293T and Mel cells, in which the silencing effect on eGFP expression was investigated by fluorescence microscopy, reverse transcription-PCR(RT-PCR), fluorescence-assited cell sorting(FACS) analysis and real-time RT-PCR. RESULTS: RNAi could effectively reduce more than 50 percent of eGFP expression in 293T cells as well as in Mel cells. CONCLUSION: The RNAi vector constructed in this way paper can effectively inhibit eGFP expression in cells.

[The effect of HSV-tk/GCV on hepatic specific damage driven by murine ALB promoter/enhancer].

The recombinant vector (pLLTK), containing murine serum albumin (ALB) gene promoter/enhancer directing the herpes simplex virus thymidine kinase (HSV-tk) gene expression was constructed to study its effect on hepatic cell-specific damage. Firstly,in order to compare the hepatic cell-specific transcriptional activity, three vectors were constructed in which green fluorescent protein (GFP) gene was used as a reporter marker. Vector pLE was driven by murine serum ALB gene promoter,whereas pLLE contained not only murine ALB promoter but also an enhancer located at upstream of the promoter was included. In the meanwhile, the vector pLEL had a murine ALB promoter and enhancer placed at the downstream from GFP. After transfected into Hep-G2 (a human hepatic cell line) and HC-11 (a murine breast epithelial cell line), GFP expression was examined by using fluorescence microscope as well as flow cytometer. Secondly, the vector pLLTK was used to observe the killing effect on Hep-G2 cells. The results demonstrated that ALB promoter/enhancer was able to direct hepatic cell-specific GFP expression. Furthermore, HSV-tk expression in Hep-G2 cells was ganciclovir (GCV)sensitive. After seven days of culture with GCV, pLLTK-transfected Hep-G2 cells showed an obvious cell death (53%) when detected by 3-4,5 dimethylthiozol-2-yl-2, 5-diphenyl tetrazolium bromide colorimetry (MTT) assay. Compared to the untreated group, there were no obvious changes in cellular growth inhibition rate in the Hep-G2 cells transfected with a blank control vector pcDNA3. 1 (only 2% of cells appeared cell death). All these results indicated that the above constructs were hepatocyte-specific. It therefore paves a way for further creating a liver-specific damage animal model by transgenic approach using HSV-tk gene expression driven by the ALB promoter/enhancer.