Construction and detection of the tissue-specific pINV-HPV16 E6/7 vector.

A tissue-specific promoter can control downstream gene expression in tissues or organs. The human involucrin (hINV) promoter (pINV) that contains 2474 bp of hINV upstream sequence is able to regulate tissue-specific gene expression. This tissue specificity may be important for the prevention and treatment of human papilloma virus infections. pINV was cloned by polymerase chain reaction and the human papillomavirus (HPV)16 E6/7 gene was obtained from the cancer tissue samples of patients with cervical carcinoma at the Yangzhou Maternal and China Health-Care Center of Jinagsu Province (Yangzhou, China). First, specific primers were designed according to the genomic DNA sequence of the HPV16-type standard strain that has been reported and the E6/7 gene was acquired by PCR. The carcinogenic fraction of the E6/7 gene was removed and the remaining section was cloned into T vectors, sequenced correctly and then cloned into the eukaryotic expression vector pCEP4, which was lacking the CMV promoter. The positive recombinants were identified using blue-white screening and endonuclease digestion, subsequent to sequencing and analysis, and the tissue-specific recombinant pINV-HPV16E6/7 plasmids was detected.

Identification of a novel mouse brachyury (T) allele causing a short tail mutation in mice.

Mutations in T-box genes are associated with numerous disease states in humans. The objective of this paper was to characterize the T(shao), a specific T-box mutation, in mice. T(shao), a short-tailed mutant mouse strain in a B6 background, was obtained by ethylnitrosourea mutagenesis. Microsatellite genomic scans mapped the location of the mutation. RT-PCR was used to amplify the identified region and the product was sequenced. DNA of the region was sequenced and scanned for mutations. Tails of T(shao) mice were mostly curly with tail length ranging from less than 1 cm (tail bud) to half of the normal length. T(shao) presented single dominance gene inheritance, and homozygous mutant mice died approximately at E10. Scans of the F2 generation mapped the mutant gene to chromosome 17, near D17Mit143. The Brachyury (T) gene was identified as a potential candidate gene in this location. To confirm this, RT-PCR was performed on RNA from intercrossed 8.5-day embryos, and products were sequenced. A 67-nucleotide deletion in exon 2 of the mutant T gene was identified. Further sequencing of the genomic DNA from this region identified a T to A transversion at the 67th nucleotide of exon 2. The T(shao) mutation is a result of a deletion in exon 2 causing the early termination and loss of function of protein encoded by the T gene, manifesting as a short tail phenotype.

[Construction and identification of recombinant adeno-associated virus expressing siRNA].

Adeno-associated virus (AAV) mediated RNA interference can be used to inhibit the expression of homologous genes in different mammalian cells. In this study, a transfer plasmid (pAAV-EGFP-H1) containing the H1 promoter and EGFP-expressing cassette was constructed based on the backbone of pAAV-MCS. Using calcium phosphate precipitation method, pAAV-EGFP-H1 was co-transfected into AAV-293 cells with helper plasmids and infective recombinant AAV was generated. EGFP gene was selected as the interfering target. EGFP gene was removed from pAAV-EGFP-H1 and a new transfer plasmid pAAV-H1 was constructed. Recombinant AAV-H1-shEGFP then infected 293 cells which was pre-transfected with plasmid pEGFP-N1. After 72 hours, the interference effect on EGFP expression was investigated by fluorescence microscope, fluorescence quantitative PCR and fluorescence activated cell sorting (FACS). All results showed that rAAV-H1-shEGFP could effectively reduce more than 60 percent of EGFP expression in 293 cells. The study demonstrates that a recombinant AAV transfer plasmid for RNAi is constructed, and the generated recombinant AAV can be used for further investigation on RNAi research.

[Construction of Fab antibody against Rh antigen].

AIM: To Construct Fab antibody against Rh antigen. METHODS: The variable regions of light and heavy chains were amplified by RT-PCR from the PBMCs of volunteers with high titer (1:256-512 by inditect agglutation) antibody to Rh antigen. Meanwhile, the genes of constant regions of light and heavy chains were isolated from pComb3xTT and pComb3xlambda phagmid carrying the templates respectively. Vkappa light chain and Fd heavy chain were linked by the first splicing overlapping extension PCR (SOE), and a full-length Fab gene was created by the second SOE. The Fab gene was ligated to phagmid pComb3HxSS and transformed to E.coli XL1-Blue by electroporation. The obtained human Fab phage antibody library was panned using Rh(-)/Rh(+) RBC four times. the phage antibodies against Rh antigen were highly enriched. Indirect agglutation test, western blot analysis and sequencing analysis were performed to detect the specificity of Fab against Rh. RESULTS: The repertoire of human phage display Fab library was 7.4 x 10(6);. After panning, A Fab clone which could bind to Rh antigen specifically was obtained. CONCLUSION: A Fab antibody that specifically aggulated Rh(+) RBC is obtained, this makes it possible to produce Rh antibody with high quantity and effection in our country.

Involvement of insulin-like growth factor-insulin receptor signal pathway in the transgenic mouse model of medulloblastoma.

A transgenic mouse model expressing Simian virus 40 T-antigen (SV40Tag) under the control of a tetracycline system was generated. In this model, a cerebellar tumor was developed after doxycycline hydrochloride treatment. Real time-polymerase chain reaction and immunohistochemistry results indicated that the SV40Tag gene was expressed in the tumor. Pathological analysis showed that the tumor belonged to medulloblastoma. Further molecular characterization of the tumor demonstrated that the insulin-like growth factor (IGF) signaling pathway was activated. We also found that the SV40Tag could bind and translocate insulin receptor substrate 1 into the nucleus in primary cultured tumor cells. The interaction between the IGF pathway and SV40Tag may contribute to the process of malignant transformation in medulloblastoma. This transgenic animal model provides an important tool for studies on the signal pathways involved in the preneoplastic process in medulloblastoma and could help to identify therapeutic targets for brain tumors.

[The effect of HPV16E7 DNA vaccine transdermal delivery with microneedle array].

OBJECTIVE: To study the effects of DNA vaccine transdermal delivery with microneedle array. METHODS: The pcDNA3.1-HPV16E7 recombinant vector acting as gene vaccine was established. The infiltration quantity of pcDNA3.1-HPV16E7 getting across the microchannels generated by microneedle arrays in vitro was observed. 30 BALB/c mice were divided into 3 groups (experimental group, in vain plasmid group, negative control). Each group had 10 mice. Then immunized BALB/c mice with a dose of 200 microg with microneedle array every two weeks. The control groups did the same as that as the study groups. Two weeks after the third immunization, the serum and lymphocytes were separated to detect the functions of humoral immunity with indirect immunofluorescence test, while, the functions of cellular immunity with lymphocyte transformation test was also detected. RESULTS: The DNA vaccine could easily get across the microchannels generated by microneedle arrays in vitro. Moreover, the course was permanent and the whole infiltration quantity was comparatively high, reaching 0.73819 mg/cm2 at the 30th hour. And among immunized BALB/c mouse, DNA vaccine transdermal delivery with microneedle array could induce specific antibodies. Lymphocyte transformation test showed that there was significant difference for the lymphocyte transformation rate between experiment (the average of lymphocyte transformation rate was 47.25%) and control group (the average of lymphocyte transformation rate was 30.00%) (chi2 = 12.903, P < 0.001). Also, the difference was found between in vain plasmid group (the average of lymphocyte transformation rate was 43.00%) and negative control(chi2 = 7.292, P = 0.007). While, no difference was observed in the experimental group and in vain plasmid group (chi2 = 0.817, P = 0.366). CONCLUSION: The DNA vaccine combined administering with microneedle array might get across the microchannels generated by microneedle arrays in vitro and induce humoral and cellular immune response in vivo.

Generation and characterization of islet cell tumor in pTet-on/pTRE-SV40Tag double-transgenic mice model.

A line of double-transgenic mice that develop neoplasms arising primarily in the pancreas was established. In these mice, the oncogene SV40 T antigen (Tag) was detected in the pancreas with and without the control of Tet-on system. The transgenic mice that developed pancreatic tumors as early as 20 weeks of age showed hypoglycemia on a blood glucose test. Pathological and immunohistochemical characterizations demonstrated that the tumors belonged to neuroendocrine neoplasms arising from pancreatic islets. A change in IGFs/IGF-1R signaling pathway was detected using real-time PCR analysis. A potential association between the IGFs/IGF-1R system and SV40Tag was studied to further explain the cancerogenesis of the double-transgenic mice by Western blot analysis and immunoprecipitation experiments. The results suggest that a Tag transgenic mice model could be used to study the molecular mechanism of the tumorigenesis of islets.

[Preparation and identification of monoclonal antibodies against Type M/N and Glycophorin A/B].

AIM: To prepare monoclonal antibodies(mAbs) against Type M/N and monoclonal antibodies against Glycophorin A and Glycophorin B (GPA/GPB) and identify rare blood group MkMk. METHODS: 8 week-old female BALB/c mice were immunized with type "O" red blood cells, Splenocytes from the immunized mice were fused with Sp2/0 myeloma cells, and positive hybridoma clones were screened by panel erythrocytes. The titer of supernatant and ascitic fluid was tested by direct and indirect agglutination. The subclasses and isotypes were identified by monoclonal antibody isotyping kit. The complemental structures of the combining sites of their antibody antigen were determined by enzyme-treated red cells. The specificity of the GPA/GPB mAbs was identified by Western blot analysis. RESULTS: Eight hybridoma cell lines against Type M/N and GPA/GPB were obtained. The titer of supernatant was between 1 x 2(-4) - 1 x 2(-8), and the titer of ascitic fluid was between 1 x 2(-7) - 1 x 2(-12). The immunoglobulin subclasses of all the mAbs were IgG except 1C1C9C4, which was IgM. Four anti-M mAbs and one anti-N mAb were deterimined by agglutination test using panel erythrocytes and the test of the combining sites in antiody antigen. Western blot analysis proved that three mAbs recognized GPA/GPB protein. CONCLUSION: Eight hybridoma cell lines against Type M/N and GPA/GPB have been obtained successfully. Among them, four mAbs recognize M, one recognizes N and three recognize GPA/GPB. Our study may be useful to research into MN blood group and for serological diagnosis. The anti-GPA/GPB mAbs can be used to prepare diabodies for the treatment and diagnosis of some of diseases.

Gene expression analysis of pancreatic cystic neoplasm in SV40Tag transgenic mice model.

AIM: To study the gene expression changes in pancreatic cystic neoplasm in SV40Tag transgenic mice model and to provide information about the prevention, clinical diagnosis and therapy of pancreatic cancer. METHODS: Using the pBC-SV40Tag transgenic mice model of pancreatic cystic neoplasm, we studied the gene expression changes by applying high-density microarrays. Validation of part gene expression profiling data was performed using real-time PCR. RESULTS: By using high-density oligonucleotide microarray, of 14113 genes, 453 were increased and 760 decreased in pancreatic cystic neoplasm, including oncogenes, cell-cycle-related genes, signal transduction-related genes, skeleton-related genes and metabolism-related genes. Among these, we confirmed the changes in Igf, Shh and Wnt signal pathways with real-time PCR. The results of real-time PCR showed similar expression changes in gene chip. CONCLUSION: all the altered expression genes are associated with cell cycle, DNA damage and repair, signal pathway, and metabolism. SV40Tag may cooperate with several proteins in promoting tumorigenesis.

Generation and characterization of a transgenic mouse model for pancreatic cancer.

AIM: To generate a SV40Tag transgenic tumor animal model and to study the mechanism underlying tumorigenesis. METHODS: A mammary gland expression vector containing SV40Tag DNA was generated. Transgene fragments were microinjeted into fertilized eggs of FVB mice. The genetically manipulated embryos were transferred into the oviducts of pseudo-pregnant female mice. PCR and Northern blot analysis were used for genotype analysis of F1 and F2 mice. Transgene expression was detected by RT-PCR and immunohistochemistry. RESULTS: SV40Tag gene was detected in two lines of transgenic mice. One of them delivered the transgene to F1 and a tumor was found in the pancreas of these mice. RT-PCR and immunohistochemistry showed that SV40Tag gene was expressed in the tumor. Pathological characterization of the transgenic mice demonstrated that the tumor belonged to pancreatic cystic neoplasm. CONCLUSION: SV40Tag transgenic mouse model can be successfully established. The transgenic mice develop a pancreatic tumor, which can be used for investigation of the molecular mechanism of tumorigenesis in vivo.

Two kinds of ENU-induced scant hair mice and mapping of the mutant genes.

OBJECTIVE: To establish mouse models for human diseases through N-ethyl-N-nitrosourea (ENU) mutagenesis, and to provide groundwork to clone genes and study their functions after mapping the mutant genes. METHODS: 18 male D2 mice (G0) at age of 8-10 weeks old were injected intraperitoneally with ENU (100 mg/kg) once a week for three consecutive weeks. The treated male mice were mated with females of the same strain, and their offspring (G1) were used to screen for dominant and recessive mutation. After breeding the mutant F2 (D2B6 F1 intercrossing) mice, 39 microsatellites that are equally distributed on the mouse genome and are different between B6 and D2 strains were used to scan the genome. According to the log odds score (LODS) we determined whether these microsatellites were linked to the mutant genes and calculated the location of mutant genes based on their recombination ratio. RESULTS: We screened 532 G1 mice, of which 14 exhibited mutation phenotypes. None was dominantly hereditable. Two cases of recessive inheritable scant hair mice were obtained through testing 30 G1 mice with normal phenotype and potential recessive mutant genes. All showed scant coat hair, grew slowly, and hyperkeratoses of epidermis and bollicular horn plug in histological sections. Their visceral organs were not markedly different from normal, and they were named scant hair 1 Baojin (symbol is snthr(-1Bao)) and scant hair 2 Baojin (symbol is snthr(-2Bao)). Through microsatellite screening we found that the LODS between snthr(-1Bao) and D9Mit243 was 7.73, and the linkage was determined. After analyzing the recombination ratio between snthr(-1Bao) and microsatellite D9Mit18 which was near snthr(-1Bao) based on a total number of 126 F2 mice with the scant hair phenotype, we determined that snthr(-1Bao) was located at chromosome 9 and was 71cM from centromere. Using the same technique, snthr(-2Bao) was mapped to the same position as snthr(-1Bao). CONCLUSION: In our research, two cases of scant hair mice provide good models for the study of dermatology, and the location of mutant genes provides a solid foundation for cloning new mice scant hair genes.

Preliminary study on the production of transgenic mice harboring hepatitis B virus X gene.

AIM:To establish transgenic mice lineage harboring hepatitis B virus X gene and to provide an efficient animal model for studying the exact role of the HBx gene in the process of hepatocarcinogenesis.METHODS:The HBx transgenic mice were produced by microinjecting the construct with X gene of HBV (subtype adr) DNA fragment into fertilzed eggs derived from inbred C57BL/6 strain; transgenic mice were identified by using Nested PCR; expression and phenotype of HBx gene were analyzed in liver from transgenic mic at the age of 8 weeks by RT-PCR, pathologic examination and periodic acid-schiff staining (PAS), respectively.RESULTS:Five hundred and fourteen fertilized eggs of C57 BL/6 mice were microinjected with recombinant retroviral DNA fragment, and 368 survival eggs injected were transferred to the oviducts of 18 pseudopregnant recipient mice, 8 of them became pregnant and gave birth to 20 F1 offspring. Of 20 offsprings, four males and two females carried the hybrid gene (HBx gene). Four male mice were determined as founder, named X 1, X 5, X 9 and X 15. These founders were back crossed to set up F1 generations with other ibred C57BL/6 mice or transgenic littermates, respectively.Transmission of HBx gene in F1 offspring of X 1, X 5 and X 9 except in X 15 followed Mendelian rules. The expression of HBx mRNA was detected in liver of F1 offspring from the founder mice (X 1 and X 9), which showed vacuolation lesion and glycogen positive foci.CONCLUSION:Transgenic mice harboring HBx gene were preliminarily established.