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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.
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Distrofia Muscular do Cíngulo dos Membros , Mutação de Sentido Incorreto , Humanos , Animais , Camundongos , Proteínas Musculares/genética , Músculo Esquelético/patologia , Distrofia Muscular do Cíngulo dos Membros/genética , Mutação , Calpaína/genética , Modelos Animais de DoençasRESUMO
Mental retardation is the main clinical manifestation of Down syndrome (DS), and neural abnormalities occur during the early embryonic period and continue throughout life. Tc1, a model mouse for DS, carries the majority part of the human chromosome 21 and has multiple neuropathy phenotypes similar to patients with DS. To explore the mechanism of early neural abnormalities of Tc1 mouse, induced pluripotent stem (iPS) cells from Tc1 mice were obtained, and genome-wide gene expression and methylation analysis were performed for Tc1 and wild-type iPS cells. Our results showed hypermethylation profiles for Tc1 iPS cells, and the abnormal genes were shown to be related to neurodevelopment and distributed on multiple chromosomes. In addition, important genes involved in neurogenesis and neurodevelopment were shown to be downregulated in Tc1 iPS cells. In short, our study indicated that genome-wide hypermethylation leads to the disordered expression of genes associated with neurodevelopment in Tc1 mice during early development. Overall, our work provided a useful reference for the study of the molecular mechanism of nervous system abnormalities in DS.
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Síndrome de Down/genética , Neurogênese/genética , Animais , Células Cultivadas , Metilação de DNA , Modelos Animais de Doenças , Humanos , Células-Tronco Pluripotentes Induzidas , CamundongosRESUMO
Colorectal cancer (CRC), one of the most malignant cancers, is currently the fourth leading cause of cancer deaths worldwide. Recent studies indicated that long non-coding RNAs (lncRNAs) could be robust molecular prognostic biomarkers that can refine the conventional tumor-node-metastasis staging system to predict the outcomes of CRC patients. In this study, the lncRNA expression profiles were analyzed in five datasets (GSE24549, GSE24550, GSE35834, GSE50421, and GSE31737) by probe set reannotation and an lncRNA classification pipeline. Twenty-five lncRNAs were differentially expressed between CRC tissue and tumor-adjacent normal tissue samples. In these 25 lncRNAs, patients with higher expression of LINC01296, LINC00152, and FIRRE showed significantly better overall survival than those with lower expression (P < 0.05), suggesting that these lncRNAs might be associated with prognosis. Multivariate analysis indicated that LINC01296 overexpression was an independent predictor for patients' prognosis in the test datasets (GSE24549, GSE24550) (P = 0.001) and an independent validation series (GSE39582) (P = 0.027). Our results suggest that LINC01296 could be a novel prognosis biomarker for the diagnosis of CRC.
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Biomarcadores Tumorais/biossíntese , Neoplasias Colorretais/genética , Prognóstico , RNA Longo não Codificante/biossíntese , RNA Longo não Codificante/genética , Idoso , Biomarcadores Tumorais/genética , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-IdadeRESUMO
Vasovagal syncope (VVS) causes accidental harm for susceptible patients. However, pathophysiology of this disorder remains largely unknown. In an effort to understanding of molecular mechanism for VVS, genome-wide gene expression profiling analyses were performed on VVS patients at syncope state. A total of 66 Type 1 VVS child patients and the same number healthy controls were enrolled in this study. Peripheral blood RNAs were isolated from all subjects, of which 10 RNA samples were randomly selected from each groups for gene expression profile analysis using Gene ST 1.0 arrays (Affymetrix). The results revealed that 103 genes were differently expressed between the patients and controls. Significantly, two G-proteins related genes, GPR174 and GNG2 that have not been related to VVS were among the differently expressed genes. The microarray results were confirmed by qRT-PCR in all the tested individuals. Ingenuity pathway analysis and gene ontology annotation study showed that the differently expressed genes are associated with stress response and apoptosis, suggesting that the alteration of some gene expression including G-proteins related genes is associated with VVS. This study provides new insight into the molecular mechanism of VVS and would be helpful to further identify new molecular biomarkers for the disease.
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Proteínas de Ligação ao GTP/genética , Receptores Acoplados a Proteínas G/genética , Síncope Vasovagal/genética , Adolescente , Estudos de Casos e Controles , Criança , Feminino , Perfilação da Expressão Gênica/métodos , Predisposição Genética para Doença , Humanos , Masculino , Análise em Microsséries/métodos , Reação em Cadeia da Polimerase em Tempo Real , Síncope Vasovagal/fisiopatologiaRESUMO
Introduction: Down syndrome (DS) is the most common genetic condition that causes intellectual disability in humans. The molecular mechanisms behind the DS phenotype remain unclear. Therefore, in this study, we present new findings on its molecular mechanisms through single-cell RNA sequencing. Methods: Induced pluripotent stem cells (iPSCs) from the patients with DS and the normal control (NC) patients were differentiated into iPSCs-derived neural stem cells (NSCs). Single-cell RNA sequencing was performed to achieve a comprehensive single-cell level differentiation roadmap for DS-iPSCs. Biological experiments were also performed to validate the findings. Results and Discussion: The results demonstrated that iPSCs can differentiate into NSCs in both DS and NC samples. Furthermore, 19,422 cells were obtained from iPSC samples (8,500 cells for DS and 10,922 cells for the NC) and 16,506 cells from NSC samples (7,182 cells for DS and 9,324 cells for the NC), which had differentiated from the iPSCs. A cluster of DS-iPSCs, named DS-iPSCs-not differentiated (DSi-PSCs-ND), which had abnormal expression patterns compared with NC-iPSCs, were demonstrated to be unable to differentiate into DS-NSCs. Further analysis of the differentially expressed genes revealed that inhibitor of differentiation family (ID family) members, which exhibited abnormal expression patterns throughout the differentiation process from DS-iPSCs to DS-NSCs, may potentially have contributed to the neural differentiation of DS-iPSCs. Moreover, abnormal differentiation fate was observed in DS-NSCs, which resulted in the increased differentiation of glial cells, such as astrocytes, but decreased differentiation into neuronal cells. Furthermore, functional analysis demonstrated that DS-NSCs and DS-NPCs had disorders in axon and visual system development. The present study provided a new insight into the pathogenesis of DS.
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BACKGROUND: Chromosome abnormalities, especially trisomy of chromosome 21, 13, or 18 as well as sex chromosome aneuploidy, are a well-established cause of pregnancy loss. Cultured cell karyotype analysis and FISH have been considered reliable detectors of fetal abnormality. However, results are usually not available for 3-4 days or more. Multiplex ligation-dependent probe amplification (MLPA) has emerged as an alternative rapid technique for detection of chromosome aneuploidies. However, conventional MLPA does not allow for relative quantification of more than 50 different target sequences in one reaction and does not detect mosaic trisomy. A multiplexed MLPA with more sensitive detection would be useful for fetal genetic screening. METHODS: We developed a method of array-based MLPA to rapidly screen for common aneuploidies. We designed 116 universal tag-probes covering chromosomes 13, 18, 21, X, and Y, and 8 control autosomal genes. We performed MLPA and hybridized the products on a 4-well flow-through microarray system. We determined chromosome copy numbers by analyzing the relative signals of the chromosome-specific probes. RESULTS: In a blind study of 161 peripheral blood and 12 amniotic fluid samples previously karyotyped, 169 of 173 (97.7%) including all the amniotic fluid samples were correctly identified by array-MLPA. Furthermore, we detected two chromosome X monosomy mosaic cases in which the mosaism rates estimated by array-MLPA were basically consistent with the results from karyotyping. Additionally, we identified five Y chromosome abnormalities in which G-banding could not distinguish their origins for four of the five cases. CONCLUSIONS: Our study demonstrates the successful application and strong potential of array-MLPA in clinical diagnosis and prenatal testing for rapid and sensitive chromosomal aneuploidy screening. Furthermore, we have developed a simple and rapid procedure for screening copy numbers on chromosomes 13, 18, 21, X, and Y using array-MLPA.
Assuntos
Aneuploidia , Técnicas de Amplificação de Ácido Nucleico/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Cromossomos Humanos Par 13 , Cromossomos Humanos Par 18 , Cromossomos Humanos Par 21 , Cromossomos Humanos X , Cromossomos Humanos Y , Sondas de DNA , Feminino , Humanos , Cariotipagem , Masculino , Mosaicismo , GravidezRESUMO
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.
Assuntos
Deleção de Genes , Duplicação Gênica , Testes Genéticos/métodos , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Feminino , Humanos , MasculinoRESUMO
microRNA (miRNA) is a class of non-coding small RNA molecules with roughly 22 nucleotides in length, regulating gene expression on post-transcriptional level and playing an important role in cell proliferation, differentiation and apoptosis process. Based on the conservation of miRNAs sequence, we compared the known miRNAs among five mammals, i.e., human, mouse, cattle, pig and dog with the sequence of sheep genome that is highly homologous to goat genome, published on the NCBI, and 11 candidate miRNAs were eventually obtained. RT-PCR analysis showed the expression of the 11 miRNAs in brain and 5 in liver, indicating that they might be novel miRNAs. The methodology provides an alternative approach to the exploration of new miRNAs in goat.
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Cabras , MicroRNAs , Animais , Biologia Computacional , Cabras/genética , Humanos , MicroRNAs/genéticaRESUMO
To explore the feasibility and accuracy of MLPA-based array (Array-MLPA) in detecting sex chromosome abnormalities, MLPA probes were designed to target against three gene loci, TSPY (p11.2), PRY (q11), and RBMY (q11.2) in human Y chromosome. Array-MLPA approach was applied to test abnormalities of Y chromosome in 15 patient samples with known karyotypes. The data were compared with karyotyping and PCR analyses. The results showed that the copy number of each site detected by Array-MLPA was basically consistent with karyotyping analysis. Moreover, small deletions of chromosomes that were not found by routine karyotyping analysis were identified by the approach described, which fully agreed with PCR analysis, indicating that Array-MLPA was able to detect small abnormalities of chromosomes that cannot be found by karyotyping analysis. Compared to the routine karyotyping method, Array-MLPA has the advantages of high efficiency and reliability in chromosomal analysis, which has great potential in clinical application of diagnosis of chromosome abnormalities.
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Aberrações Cromossômicas , Cromossomos Humanos Y/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Humanos , Cariotipagem , Reação em Cadeia da PolimeraseRESUMO
Trisomy 21 is the most common chromosomal disorder and underlies Down syndrome. Epigenetics, such as DNA methylation and post-translational histone modifications, plays a vital role in Down syndrome. However, the functions of epigenetics-related long noncoding RNAs (lncRNAs), found to have an impact on neural diseases such as Alzheimer's disease, remain unknown in Down syndrome. In this study, we analyzed the RNA sequencing data from Down syndrome-induced pluripotent stem cells (iPSCs) and normal iPSCs. A large number of lncRNAs were identified differentially expressed in Down syndrome-iPSCs. Notably, stronger perturbation was shown in the expression of lncRNAs compared to protein coding genes (Kolmogorov-Smirnov test, P<0.05), suggesting that lncRNAs play more important roles in Down syndrome. Through gene set enrichment analysis and bi-clustering, we also found that most of the differential expressed lncRNAs were closely associated with mitochondrial functions (e.g. mitochondrion organization, P=3.21×10-17; mitochondrial ATP synthesis coupled electron transport, P=1.73×10-19 and mitochondrial membrane organization, P=4.04×10-8). PCR-array and qRT-PCR results revealed that almost all genes related to mitochondria were down-regulated in Down syndrome-iPSCs, implying that mitochondria were dysfunctional in Down syndrome (e.g. ATP5B, Fold Change=-8.2317; COX6A1, Fold Change=-12.7788 and SLC25A17, Fold Change=-22.1296). All in all, our study indicated that a stronger perturbation of lncRNAs expression may lead to the dysfunction of mitochondria in Down syndrome.
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Síndrome de Down/genética , Síndrome de Down/patologia , Perfilação da Expressão Gênica , Mitocôndrias/genética , RNA Longo não Codificante/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismoRESUMO
The discovery of cell-free DNA fetal (cff DNA) in maternal plasma during pregnancy provides a novel perspective for the development of noninvasive prenatal diagnosis (NIPD). Against the background of maternal DNA, the use of the relatively low concentration of cff DNA is limited in NIPD. Therefore, in order to overcome the complication of the background of maternal DNA and expand the scope of cff DNA application in clinical practice, it is necessary to identify novel universal fetalspecific DNA markers. The GeneChip Human Promoter 1.0R Array set was used in the present study to analyze the methylation status of 12 placental tissue and maternal peripheral blood wholegenome DNA samples. In total, 5 fetus differential hypermethylation regions and 6 fetus differential hypomethylation regions were identified. In order to verify the 11 selected methylation regions and detect the differential CpG sites in these regions, a bisulfate direct sequencing strategy was used. In total, 87 fetal differential methylation CpG sites were identified from 123 CpG sites. The detection of fetal differential methylation DNA regions and CpG sites may be instrumental in the development of efficient NIPD and in the expansion of its application in other disorders.
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Metilação de DNA , Epigênese Genética , Epigenômica , Estudo de Associação Genômica Ampla , Diagnóstico Pré-Natal , Adulto , Biomarcadores , Biologia Computacional/métodos , Ilhas de CpG , Epigenômica/métodos , Feminino , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla/métodos , Idade Gestacional , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Gravidez , Diagnóstico Pré-Natal/métodos , Regiões Promotoras Genéticas , Análise de Sequência de DNARESUMO
Epigenetics regulations have an important role in fertilization and proper embryonic development, and several human diseases are associated with epigenetic modification disorders, such as Rett syndrome, Beckwith-Wiedemann syndrome and Angelman syndrome. However, the dynamics and functions of long non-coding RNAs (lncRNAs), one type of epigenetic regulators, in human pre-implantation development have not yet been demonstrated. In this study, a comprehensive analysis of human and mouse early-stage embryonic lncRNAs was performed based on public single-cell RNA sequencing data. Expression profile analysis revealed that lncRNAs are expressed in a developmental stage-specific manner during human early-stage embryonic development, whereas a more temporal-specific expression pattern was identified in mouse embryos. Weighted gene co-expression network analysis suggested that lncRNAs involved in human early-stage embryonic development are associated with several important functions and processes, such as oocyte maturation, zygotic genome activation and mitochondrial functions. We also found that the network of lncRNAs involved in zygotic genome activation was highly preservative between human and mouse embryos, whereas in other stages no strong correlation between human and mouse embryo was observed. This study provides insight into the molecular mechanism underlying lncRNA involvement in human pre-implantation embryonic development.
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Regulação da Expressão Gênica no Desenvolvimento , RNA Longo não Codificante/genética , Transcriptoma , Animais , Blastocisto/metabolismo , Ciclo Celular , Desenvolvimento Embrionário/genética , Epigênese Genética , Perfilação da Expressão Gênica , Genoma , Genoma Humano , Humanos , Camundongos , Modelos Estatísticos , Mórula/metabolismo , Oócitos/metabolismo , Curva ROC , Análise de Sequência de RNA , Análise de Célula Única , Fatores de Tempo , Zigoto/metabolismoRESUMO
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.
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Cabras/embriologia , Modelos Animais , Transplante de Células-Tronco/métodos , Ultrassonografia/métodos , Animais , Cromossomos Humanos Par 17/genética , DNA/sangue , DNA Satélite/sangue , Feminino , Citometria de Fluxo , Idade Gestacional , Glicoforinas/genética , Cabras/sangue , Humanos , Hibridização in Situ Fluorescente , Cavidade Peritoneal/embriologia , Reação em Cadeia da Polimerase , Gravidez , Quimeras de Transplante/genética , Transplante HeterólogoRESUMO
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.
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Transplante de Células-Tronco Hematopoéticas , Animais , Feminino , Citometria de Fluxo , Hibridização in Situ Fluorescente , Camundongos , Modelos Animais , Reação em Cadeia da Polimerase , Quimeras de Transplante , Transplante HomólogoRESUMO
Hemophilia B is an X-linked bleeding disease, caused by the mutations of human coagulation factor IX (hFIX) gene located in chromosome X. It results in a dramatic decline of hFIX quantity or clotting activity in plasma, and the intrinsic clotting pathway is affected seriously. In this article, the structure and function of hFIX gene as well as the protein encoded by this gene were reviewed. Various types of hFIX mutants found in hemophilia B were also described, including the mutations caused by founder effects, mutations in regulatory region, coding region, splicing sites of introns and two other special mutations. Meanwhile, the biological effects of the mutations described above were discussed. Finally, a mutation type (Arg-->Ala at point 338) that can increase the clotting activity of hFIX as well as the potential application was briefly introduced.
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Fator IX/genética , Efeito Fundador , Hemofilia B/genética , Mutação , Animais , Coagulação Sanguínea/genética , Coagulação Sanguínea/fisiologia , Fator IX/fisiologia , Hemofilia B/sangue , Hemofilia B/fisiopatologia , Humanos , Regiões Promotoras Genéticas/genética , Splicing de RNARESUMO
In this report, a full-length sequence of bovine prolactin (bPRL) genomic DNA with 9388 bp, which has been accepted by GenBank (Accession Number: AF426315), was firstly cloned by Long PCR procedures. This sequence consists of 5 exons, 4 introns, 854 bp of 5' upstream regulatory region and 69 bp of 3' UTR. Accession number of protein encoded by AF426315 sequence in GenBank is AAL28075 that is composed of 229 amino acid residues, in which signal sequence resides in 1-30 sites and mature polypeptide consists of 199 amino acid residues. The recombinant plasmid containing bPRL genomic DNA was then transfected into eukaryotic cells (COS-7), followed by RT-PCR procedure. An 804 bp of bPRL cDNA containing all the encoding region was obtained, indicating that the bPRL genomic DNA reported herein had its biological function at the transcriptional level. Results derived from information searching by Blast program revealed that there were various SNP sites in the sequences of bPRL mRNAs and ESTs collected in GenBank, which located mainly in downstream encoding region and 3' UTR. These SNP sites did not alter the related amino acids encoded. In addition, mRNA sequences encoding 5' signal sequence of bPRL gene was highly conserve.
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Bovinos/genética , Prolactina/genética , Animais , Células COS , Clonagem Molecular , DNA/química , DNA/genética , DNA Complementar/química , DNA Complementar/genética , Dados de Sequência Molecular , Filogenia , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNARESUMO
In utero stem cells transplantation is a promising approach to the prenatal treatment of diseases. In order to investigate the fate of the stem cells after in utero transplantation, we have established a chimeric mouse model with the method of in utero transplantation. Mononuclear cells (including stem cells/progenitor cells) derived from male mouse bone marrow were injected into fetal mouse peritoneal cavity during the pre-immune period. The donor cells in the circulatory blood of female recipients were identified by fluorescence in situ hybridization (FISH), and the Y-chromosome specific DNA was detected by PCR as well as real-time quantitative PCR after the recipient mice were born. The results showed that a total of 4 female recipient mice were chimeric in their peripheral blood. Significantly, the donor cells in three chimeric mice persisted up to six months.
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Feto/cirurgia , Transplante de Células-Tronco Hematopoéticas/métodos , Quimeras de Transplante/sangue , Animais , Animais Recém-Nascidos , Feminino , Hibridização in Situ Fluorescente , Masculino , Camundongos , Modelos Animais , Gravidez , Resultado da Gravidez , Quimeras de Transplante/genética , Transplante Homólogo , DesmameRESUMO
To probe the feasibility of efficient production of human clotting factor IX(hFIX) with the approach of mammary gland bioreactor of transgenic animals, we constructed hFIX mammary gland expression vector containing promoter, exon 1, intron 1 and exon 2 of the goat beta-casein gene about 6.7 kb fragment as well as full-length of hFIX cDNA and its modified intron 1 sequence. By using transgenic products and 12 transgenic founders (9 female, 3 male) were produced, and the integration rate thus was 11.2%. ELISA assay and Western blot showed that the milk of 8 female transgenic mice had hFIX expression with high clotting activities. The highest hFIX expression in the milk of one transgenic mouse reached 52.9 mg/L, and the highest clotting activity of the transgene milk was 279.2%. FISH experiments indicated that hFIX DNA was integrated in different chromosomes in different mice. This result indicated that the hFIX mammary gland expression vector based on the goat beta-casein promoter can efficiently direct high expression of hFIX gene in the milk of transgenic mice, which maintained high clotting activity.
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Caseínas/genética , Fator IX/genética , Regiões Promotoras Genéticas , Animais , Western Blotting , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Cabras , Humanos , Hibridização in Situ Fluorescente , Camundongos , Camundongos TransgênicosRESUMO
To clone goat beta-lactoglobulin (BLG) gene,two fragments were amplified from goat genomic DNA by LD-PCR method. The fragments were inserted in T-vectors before being spliced into the whole 7.2 kb BLG gene at a single restriction enzyme site of NarI. Consequently, the eukaryotic expression vector was constructed. All the clones were proved to be correct by restriction enzyme cutting and sequencing analysis. Six Founders (3 male symbol, 3 female symbol) of goat BLG transgenic mice were obtained by microinjection and BLG genes integration were confirmed by both PCR and Southern blot analyses. The milk was collected from two lactating female transgenic mice and goat BLG protein contents were measured with ELISA. The results showed that goat BLG protein in milk of the two mice were 23.49 mg/mL and 2.19 mg/mL, respectively.
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To investigate the feasibility in the promotion of the expression of foreign proteins, such as human lactinferrin (hLF) and thrombopoietin (TPO), in the transgenic animals-mammary gland bioreactor by bovine prolactin (bPRL), two types of mammary gland dual-expression vectors containing bPRL and foreign DNAs were constructed. In one type of vector, both foreign and bPRL DNAs were linked by internal ribosome entry site (IRES) which shared one goat 6.7 kb of beta-casein promoter; while in the other type of vector, the transcriptions of the foreign gene as well as bPRL were respectively directed by 6.7 kb goat beta-casein promoter and 2.0 kb of goat beta-casein promoter. After transfection of the vectors with lipofectin method, the expression of foreign proteins were measured by RT-PCR as well as ELISA assay. The results showed that bPRL could obviously promote the expression of foreign proteins (hLF and TPO) in cultured cells. The hLF expression level was increased from 12.6 microg/L to 18.4 microg/L and 37.2 microg/L in the COS-7 cells (African green monkey kidney cell) (P<0.05), and from 13.7 microg/L to 20.7 microg/L and 19.9 microg/L in the HC-11 cells (epithelial cell of mouse mammary gland) (P<0.05) by two vectors, respectively. TPO expression level was increased from 572 ng/L to 1340 ng/L in the COS-7 cell (P<0.05), and from 783 ng/L to 1040 ng/L in the HC-11 cell (P<0.05) by the vector which have two promoters. This work indicate that bovine prolactin can effectively increase the expression of foreign genes in mammal cells.