ABSTRACT
The study of human new gene's function has become an increasingly active academic field basically relying on the gene knockout (KO) mouse. The construction of targeting vector economically and efficiently has turned into the key step to acquire a KO mouse because of the low efficiency of recombination with traditional constructed targeting vector. For study of the function of new gene-Resp18, we brought in a new DNA engineering platform-Red/ET recombination to construct Resp18 targeting vector. Red/ET recombineering differs from the conventional ways of vector construction (e.g., PCR, restriction enzyme digestion and ligation) and achieves genetic modification by acquisition, insertion, fusion or replacement of the target gene through small fragments mediated homologous recombination. Now Resp18 targeting vectors of three strategies were yielded successfully through two homologous recombination processes of retrieve and neo-targeting. Red/ET recombination has the advantage of getting longer homology regions without mutation, which makes it a new and reliable alternative to the construction of a targeting vector today.
Subject(s)
Bacteriophage lambda , Genetics , Chromosomes, Artificial, Bacterial , Genetics , Gene Targeting , Methods , Genetic Engineering , Methods , Genetic Vectors , Genetics , Plasmids , Genetics , Viral Proteins , GeneticsABSTRACT
Objective To study the regulatory effect of interferons(IFNs) on retinoic acid-inducible gene-Ⅰ(RIG-Ⅰ) and the roles of RIG-Ⅰ in IFNs signaling pathway. Methods RIG-Ⅰ expression before and after IFNs treatment in mouse embryonic fibroblasts(MEFs) were analyzed with Northern blotting and semi-quantitative RT-PCR assay.MEFs isolated from wild-type and RIG-Ⅰ-/-mice were used to test growth inhibition and antiviral activity of IFNs with MTT assay and cytopathic effect inhibition assay. Results Both IFN-? and IFN-? could induce RIG-Ⅰ expression in MEFs.Treated with 100 U/mL IFN-?,growth inhibition and antiviral activity of MEFs from wild-type mice were more significant than those from RIG-Ⅰ-/-mice.With the absence of RIG-Ⅰ,the antiviral protective role IFN-? plays was significantly weaker than the wild type. Conclusion RIG-Ⅰ gene is a novel mediator of interferon effects on cells.It may participate in the inflammation responses mediated by IFNs through modulating cytokines production.
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Objective To explore the biological functions of retinoic acid-inducible gene-I(RIG-I) in vivo through phenotype analysis of RIG-I knockout mice. Methods The gene expression of RIG-Ⅰ in various tissues of mice was examined with Northern blotting and semi-quantitative RT-PCR.The phenotypes observed included body weight measurement,differential count of peripheral blood cells,metabolic parameters measurement and histopathologic examination. ResultsRIG-Ⅰ expressed in various tissues of mice with different levels.No gross developmental abnormalities and expected maturation arrest in granulocytic differentiation were observed in RIG-Ⅰ knockout mice.However,RIG-Ⅰ knockout mice exhibited an unexpected increase in the ratios of neutrophiles to lymphocytes in peripheral blood and increased susceptibility to bacteria infection. Conclusion RIG-Ⅰ may play an important role in immune regulation in mice.
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<p><b>OBJECTIVE</b>To construct the doxycycline-inducible MT transgenic mice model, and provide a basis for the study of hemangioma as well as MT molecular function in vivo.</p><p><b>METHODS</b>Tetracycline-controlled expression systems were employed to this study. A conditional transgenic vector combining the two transcriptional units on a single plasmid was constructed, and the MT gene was subcloned into this vector. To minimize any potential interference, the two elements were spaced with a 1.2 kb cHS4 insulator. To shield the transgene from the affection of chromosomal position effect and improve its expression efficiency, another cHS4 insulator was inserted into the upstream of transgene cassette. After transient transfection of cells in vitro, and analyzing the relative quantification of MT transcripts (target) in mRNA samples by semi-quantitative RT-PCR method, the pronuclear microinjection technique was used to introduce the purified transgene into the chromosomes of fertilized mice eggs, in order to obtain transgenic positive animals. The MT expression in positive mouse was induced through adding deoxycycline in drinking water. Phenotype analysis was done by pathology, and MT expression was confirmed by RT-PCR.</p><p><b>RESULTS</b>The conditional transgenic vector was constructed successfully, and the expression of MT in vitro was regulated by doxycycline. Five transgenic positive mice were obtained through pronuclear microinjection. After MT induction, one transgenic mice developed hemangiomas, and the expression of MT was confirmed by RT-PCR method. The others were active and in breeding.</p><p><b>CONCLUSION</b>Conditional MT transgenic animal model was constructed successfully, and may provide platform for the experimental research of hemangioma as well as the MT molecular function in vivo.</p>
Subject(s)
Animals , Mice , Antigens, Polyomavirus Transforming , Genetics , COS Cells , Chlorocebus aethiops , Gene Expression , Genetic Vectors , Genetics , Mice, Transgenic , Models, Genetic , Reverse Transcriptase Polymerase Chain Reaction , Tetracycline , Pharmacology , Transfection , MethodsABSTRACT
To generate transgenic mice in which both hygromycin (hyg) and neomycin (neo) resistance genes are expressed in murine fibroblast cells (MEFs), which are required for conditional gene knock-out and screening of drug resistant ES cell clones. To construct HygR-neoR expression vector, pTK-hygR-pA and PGK-neoR-pA were cloned into pBluescript vector. DNA fragments of tandem genes ( 4245bp ) were prepared by Kpn I and Xba I digestion and transgene was microinjected into pronucleus of zygotes to generate transgenic mice. Transgenic mice were identified by PCR and Southern blot; expression of hygR and neoR gene transcripts were detected by RT-PCR. 7 founder mice carrying hyg-neo resistant genes were obtained and 6 transgenic mouse lines were successfully established. The hygR and neoR gene transcripts were detected in the liver and/or ovary of transgenic mice from hn30, hn33, hn66 and hn67 mouse lines. In MEFs isolated from the mice of line hn66 and hn30, expression of hyg and neo resistant genes was also detectable. Transgenic mouse lines expressing two anti-drug genes have been established. The hyg and neo resistant gene transcripts were detected in the MEFs of two transgenic mouse lines.
Subject(s)
Animals , Mice , Cinnamates , Pharmacology , Drug Resistance, Multiple , Genetics , Fibroblasts , Metabolism , Hygromycin B , Pharmacology , Mice, Transgenic , Neomycin , Pharmacology , Transgenes , GeneticsABSTRACT
<p><b>OBJECTIVE</b>In order to investigate the leukemogenic potential of NUP98-PMX1 fusion gene in vivo.</p><p><b>METHODS</b>NUP98-PMX1 transgenic mice were generated, in which the fusion gene was driven by hCG promoter and expressed in myeloid cells at early stage of differentiation. Molecular cloning technology was used to construct NUP98-PMX1 transgenic plasmid. The genotype and phenotype of the NUP98-PMX1 transgenic mice were analyzed by PCR, RT-PCR, peripheral blood count (PBC), bone marrow (BM) cells morphology and pathological examination.</p><p><b>RESULTS</b>NIH3T3 cells transfected with NUP98-PMX1 fusion gene grew faster, formed colonies in soft agar, and developed tumors in 10 inoculated nude mice. Among 8 disordered NUP98-PMX1 transgenic mice, 4 developed myeloid leukemia-like phenotype, including 3 resembling human chronic myeloid leukemia.</p><p><b>CONCLUSION</b>NUP98-PMX1 has oncogenic activity and plays a crucial role in leukemogenesis.</p>
Subject(s)
Animals , Female , Humans , Male , Mice , Bone Marrow Cells , Metabolism , Pathology , Disease Models, Animal , Flow Cytometry , Gene Expression Regulation, Leukemic , Green Fluorescent Proteins , Genetics , Metabolism , Leukemia, Myeloid , Genetics , Pathology , Mice, Inbred C57BL , Mice, Inbred Strains , Mice, Nude , Mice, Transgenic , NIH 3T3 Cells , Nuclear Pore Complex Proteins , Genetics , Metabolism , Oncogene Proteins, Fusion , Genetics , Metabolism , Phenotype , Plasmids , Recombinant Fusion Proteins , Genetics , Metabolism , Reverse Transcriptase Polymerase Chain Reaction , TransfectionABSTRACT
<p><b>OBJECTIVE</b>In order to investigate the leukemogenic potential of NUP98-HOXA9 fusion gene in vivo.</p><p><b>METHODS</b>Molecular cloning technology was used to construct NUP98-HOXA9 transgenic plasmid and NUP98-HOXA9 transgenic mice were generated. The genotype and phenotype of the NUP98-HOXA9 transgenic mice were analyzed by PCR, RT-PCR and colony-forming assay. The effect of N-ethyl-N-nitrosourea (ENU) stimulation on the transgenic mice was analyzed by peripheral blood count, bone marrow (BM) cells morphology pathological examination.</p><p><b>RESULTS</b>The transgenic expression was detected in 5 independent lines of NUP98-HOXA9 transgenic mice, but no expected phenotypes was found in 2 year follow-up. Upon ENU stimulation, 2 of 10 transgenic mice developed myeloid leukemia, suggesting that NUP98-HOXA9 transgenic mice have increased susceptibility to ENU mutagenesis in leukemogenesis.</p><p><b>CONCLUSION</b>The fusion gene expressed in BM cells of NUP98-HOXA9 transgenic mice. It seems that the expression of the fusion gene is insufficient to trigger leukemogenesis. However, the increased susceptibility to ENU mutagenesis suggests that NUP98-HOXA9 fusion gene might play a potential role in leukemogenesis.</p>
Subject(s)
Animals , Female , Humans , Male , Mice , Bone Marrow Cells , Metabolism , Pathology , Disease Models, Animal , Ethylnitrosourea , Gene Expression Regulation, Leukemic , Genotype , Homeodomain Proteins , Genetics , Leukemia, Myeloid , Blood , Genetics , Mice, Transgenic , Nuclear Pore Complex Proteins , Genetics , Oncogene Proteins, Fusion , Genetics , Phenotype , Plasmids , Reverse Transcriptase Polymerase Chain Reaction , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To investigate the relationship between haplotypes of multilocus markers and ankylosing spondylitis (AS).</p><p><b>METHODS</b>Five families with AS were recruited from Shanghai area. Eleven microsatellite markers around D6S276 were analyzed by Linkage package and by Cyrillic package.</p><p><b>RESULTS</b>Fine linkage analysis showed the significant Lod score values with D6S276 was 3.8821, Lod score values with D6S1691 and D6S1618 near D6S276 were larger than 1.5. The crossover value in 5 pedigrees was 14%. The haplotype analysis showed that the regions between D6S1691 and D6S1618 were associated with AS.</p><p><b>CONCLUSION</b>The regions of D6S1691-D6S276-D6S1618 may harbor a susceptible gene of AS. The specific haplotypes of different pedigrees may play an important role in the presymptomatic diagnosis for AS.</p>
Subject(s)
Female , Humans , Male , Haplotypes , Genetics , Linkage Disequilibrium , Genetics , Pedigree , Spondylitis, Ankylosing , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To set up the animal model of hemangioma by microinjecting the PyMT transgenic DNA.</p><p><b>METHODS</b>Constructing the transgenic PyMT gene, and microinjecting it into fertilized embryos which were transferred to pseudopregnant recipients then. Observing the phenotype of the newborn-mice, detecting the integration of transgenic DNA by PCR, and analyzing the histological morphon of the neoplasm of the mice.</p><p><b>RESULTS</b>The transgenic DNA was proved to be right and has been microinjected into 579 fertilized embryos, 62 mice were born. Within the 62 mice, one mouse was found being the phenotype of hemangioma. PyMT gene was expressed in the total DNA of the transgenic mouse by PCR.</p><p><b>CONCLUSION</b>It could be a good way to build animal model of hemangioma with transgenic PyMT DNA.</p>
Subject(s)
Animals , Female , Male , Mice , Antigens, Polyomavirus Transforming , Genetics , Physiology , Hemangioma , Mice, Transgenic , MicroinjectionsABSTRACT
<p><b>OBJECTIVE</b>To study the biological function of fusion gene HRX-EEN and its role in leukemogenesis, and to provide an ideal animal model for anti-leukemia drug screening.</p><p><b>METHODS</b>HRX-EEN fusion gene was constructed by use of three different DNA fragments, and it was inserted into hCG transgenic vector. G(0) transgenic mice were obtained by microinjection of the recombined DNA into the pronucleus of zygotes, followed by implantation of the injected zygotes into pseudopregnant mice. The integration of the transgene was tested by PCR and its expression by reverse transcription-polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>The sequence of recombined HRX-EEN gene was confirmed by sequencing. PCR testing revealed a total of 7 G(0) transgenic mice, these mice were then mated with C57 wild type mice. Except mouse No. 35 that died, the others all had their F1 offsprings. From these 6 lines of transgenic mice, HRX-EEN gene was found to be stably expressed in 3 lines by RT-PCR. Up to now, all transgenic mice expressing the fusion gene have no obvious abnormal phenotypes.</p><p><b>CONCLUSION</b>A transgenic mice model in which the HRX-EEN fusion gene can be stably expressed has been established.</p>
Subject(s)
Animals , Mice , DNA-Binding Proteins , Genetics , Histone-Lysine N-Methyltransferase , Intracellular Signaling Peptides and Proteins , Mice, Transgenic , Myeloid-Lymphoid Leukemia Protein , Polymerase Chain Reaction , Proteins , Genetics , Proto-Oncogenes , Recombinant Fusion Proteins , Genetics , Transcription FactorsABSTRACT
<p><b>OBJECTIVE</b>Activation and overexpression of pleomorphic adenoma (PLAG1) gene due to t(3;8)(p21;q12) translocation are associated with the development of human pleomorphic adenomas of the salivary glands. This study was conducted to generate ubiquitously-expressed or tissue-specific expressed PLAG1 transgenic mice and to elucidate the role of PLAG1 gene in tumorigenesis in vivo.</p><p><b>METHODS</b>Human PLAG1 cDNA was cloned from salivary gland tumor or placenta tissues by RT-PCR. Ubiquitous expression vector pCMV-EGFP/PLAG1 driven by CMV promoter and tissue-specific expression vector pMMTV-PLAG1 driven by MMTV LTR were constructed. NIH3T3 cells transiently transfected with pCMV-EGFP/PLAG1 showed high expression of PLAG1 in nucleus. Transgenes were microinjected into pronucleus of zygotes to generate transgenic mice.</p><p><b>RESULTS</b>It was found that the human PLAG1 cDNA cloned from several salivary gland tumor and normal placenta tissues consistently showed a variation of a single nucleotide at the same position when compared with the human PLAG1 cDNA sequence in Genbank (Accession No. U65002), which led to T458P at protein level. It might be a single nucleotide polymorphism (SNP)locus. Fused EGFP/PLAG1 protein was found to be localized in the nucleus of NIH3T3 cells transiently transfected with pCMV-EGFP/ PLAG1. Several pCMV-EGFP/PLAG1 and pMMTV-PLAG1 transgenic mouse lines were obtained respectively. As might be expected, pMMTV-PLAG1 transgenic mice spontaneously developed salivary gland tumors in three independent lines, among which, line 42 showed tumorigenic phenotype in 100% of transgenic mice within three months after birth.</p><p><b>CONCLUSION</b>Overexpression of PLAG1 gene plays a crucial role in tumorigenesis of salivary gland tumors.</p>
Subject(s)
Animals , Female , Humans , Male , Mice , Base Sequence , DNA-Binding Proteins , Genetics , Disease Models, Animal , Green Fluorescent Proteins , Luminescent Proteins , Genetics , Metabolism , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Inbred Strains , Mice, Transgenic , Molecular Sequence Data , NIH 3T3 Cells , Plasmids , Genetics , Recombinant Fusion Proteins , Genetics , Metabolism , Salivary Gland Neoplasms , Genetics , Metabolism , Pathology , TransfectionABSTRACT
Six human leucocytic antigen(HLA)-associated diseases, including ankylosing spondylitis, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, type 1 diabetes mellitus and psoriasis, were selected as objects of this review. The characteristics of these diseases in whole-genome scans on susceptibility genes or loci undertaken to date were analyzed and compared. Meanwhile, the potential proposals for dealing with the existing problems were put forward.