RESUMO
MARCH5 is a ring-finger E3 ubiquitin ligase located in the outer membrane of mitochondria. A previous study has reported that MARCH5 was up-regulated and contributed to the migration and invasion of OC cells by serving as a competing endogenous RNA. However, as a mitochondrial localized E3 ubiquitin ligase, the function of MARCH5 in mitochondrial-associated metabolism reprogramming in human cancers remains largely unexplored, including OC. We first assessed the glycolysis effect of MARCH5 in OC both in vitro and in vivo. Then we analyzed the effect of MARCH5 knockdown or overexpression on respiratory activity by evaluating oxygen consumption rate, activities of OXPHOS complexes and production of ATP in OC cells with MARCH5. Co-immunoprecipitation, western-blot, and in vitro and vivo experiments were performed to investigate the molecular mechanisms underlying MARCH5-enhanced aerobic glycolysis s in OC. In this study, we demonstrate that the abnormal upregulation of MARCH5 is accompanied by significantly increased aerobic glycolysis in OC. Mechanistically, MARCH5 promotes aerobic glycolysis via ubiquitinating and degrading mitochondrial pyruvate carrier 1 (MPC1), which mediates the transport of cytosolic pyruvate into mitochondria by localizing on mitochondria outer membrane. In line with this, MPC1 expression is significantly decreased and its downregulation is closely correlated with unfavorable survival. Furthermore, in vitro and in vivo assays revealed that MARCH5 upregulation-enhanced aerobic glycolysis played a critical role in the proliferation and metastasis of OC cells. Taken together, we identify a MARCH5-regulated aerobic glycolysis mechanism by degradation of MPC1, and provide a rationale for therapeutic targeting of aerobic glycolysis via MARCH5-MPC1 axis inhibition.
Assuntos
Progressão da Doença , Glicólise , Proteínas de Transporte da Membrana Mitocondrial , Transportadores de Ácidos Monocarboxílicos , Neoplasias Ovarianas , Ubiquitina-Proteína Ligases , Ubiquitinação , Humanos , Feminino , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Animais , Linhagem Celular Tumoral , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Camundongos , Transportadores de Ácidos Monocarboxílicos/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Mitocôndrias/metabolismo , Mitocôndrias/genética , Camundongos Nus , Regulação Neoplásica da Expressão Gênica , Proliferação de Células , Camundongos Endogâmicos BALB CRESUMO
BACKGROUND: The present study aimed to determine the accuracy (Z-value) of non-invasive prenatal testing (NIPT) results for sex chromosome aneuploidy (SCA) in routine clinical practice. METHODS: Among a cohort of 12505 pregnant females, maternal plasma samples collected from our hospital were utilized for SCA analysis by NIPT detection. The positive samples were validated through an invasive procedure and karyotyping analysis. The predictive value from positive samples in sex chromosomes was compared to analyze the accuracy of the Z-value. RESULTS: There were 65 females with sex chromosome abnormalities within 12,505 pregnant females in the NIPT detection, which was validated by karyotype analysis of amniotic fluid puncture through sequencing, as well as bioinformatics analysis, with 18 true-positive samples. The true-positive results with 45,X, 47,XXY, 47,XXX and 47,XYY karyotypes predicted by NIPT were 14.29%, 50.00%, 66.67% and 71.43%, respectively. Among sex chromosome cases, the findings indicated that positive NIPT results with Z ≥ 9 show a higher accuracy. CONCLUSIONS: The findings of the present study demonstrate that the positive predictive value of NIPT for sex chromosome abnormalities is distinctive. The positive predictive value was highest for 47,XYY and lowest for 45,X. Additionally, the Z-value results are considered to be correlated with the accuracy of NIPT, although further studies need to be made.
Assuntos
Diagnóstico Pré-Natal/métodos , Aberrações dos Cromossomos Sexuais/embriologia , Transtornos dos Cromossomos Sexuais/diagnóstico , Transtornos dos Cromossomos Sexuais/genética , Cromossomos Humanos X/genética , Estudos de Coortes , Feminino , Testes Genéticos/métodos , Humanos , Cariotipagem , Síndrome de Klinefelter/diagnóstico , Síndrome de Klinefelter/genética , Valor Preditivo dos Testes , Gravidez , Aberrações dos Cromossomos Sexuais/estatística & dados numéricos , Transtornos dos Cromossomos Sexuais/sangue , Transtornos do Cromossomo Sexual no Desenvolvimento Sexual/diagnóstico , Transtornos do Cromossomo Sexual no Desenvolvimento Sexual/genética , Cromossomos Sexuais/patologia , Trissomia/diagnóstico , Trissomia/genética , Síndrome de Turner/diagnóstico , Síndrome de Turner/genética , Cariótipo XYY/diagnóstico , Cariótipo XYY/genéticaRESUMO
BACKGROUND: Central nervous system (CNS) abnormalities are a group of serious birth defects associated with high rates of stillbirths, infant death, or abnormal development, and various disease-causing copy number variations play a much more important role in the etiology of CNS abnormalities. This study intends to present a retrospective study of the prenatal diagnosis and the pregnancy outcome of fetuses diagnosed with CNS abnormalities, and evaluate the clinical value of chromosomal microarray analysis (CMA) in prenatal diagnosis of CNS abnormalities. METHODS: A total of 356 fetuses with CNS abnormalities with or without other ultrasound abnormalities subjected to invasive prenatal diagnosis at the first affiliated hospital of Air Force Medical University from January 2015 to August 2018. All cases have performed both karyotyping and CMA concurrently, but 20 fetuses with chromosome aneuploidy were excluded in the current study. RESULTS: The CMA identified pathogenic copy number variants (pCNVs) in 27/336 (8.03%) fetuses, likely pCNVs in 8/336 (2.38%) fetuses, and variants of unknown significance (VOUS) in 11/336 (3.27%) fetuses. A total of 222 cases had single CNS abnormalities and the pCNVs detection rate was 5.86% (13/222), the remaining 114 cases including CNS abnormalities plus other structural abnormalities, ultrasonographic soft markers and two or more CNS abnormalities, the pCNVs detection rate was 12.3% (14/114). CONCLUSIONS: Fetuses with CNS abnormalities have a higher risk of chromosomal abnormalities, our study showed that CNVs play an important role in the etiology of CNS abnormalities. The application of CMA could increase the detection rate of pCNVs causing CNS abnormalities.
Assuntos
Sistema Nervoso Central/anormalidades , Aberrações Cromossômicas , Análise em Microsséries , Diagnóstico Pré-Natal , Variações do Número de Cópias de DNA/genética , Feto/anormalidades , Seguimentos , Humanos , CariotipagemRESUMO
Objective: There is scant information available about fetuses with 7q11.23 copy number variants (CNVs) found during pregnancy. We studied the clinical significance of 7q11.23 CNVs in prenatal diagnosis. Materials and methods: The amniocentesis was performed on pregnant women who underwent ultrasound (US) of fetal abnormalities. After karyotype analysis, CNVs were detected using BACs-on-Beads (BoBs) technique and chromosome microarray analysis (CMA). Results: Of seven fetuses with CNV of 7q11.23, five had microdeletions and two had microduplications. Case 1 had a 7q11.23 microdeletion along with other CNVs. Case 7 was a newborn with a normal phenotype and 7q11.23 microduplication. Conclusion: The CNVs in 7q11.23 results in many clinical manifestations, but the specificity of clinical features is not high. This study demonstrated that BoBs combined with CMA allows prenatal diagnosis of CNVs involving 7q11.23, and provide a clinical basis for prenatal diagnosis and genetic counseling of such CNVs.
Assuntos
Transtornos Cromossômicos , Amniocentese , Aberrações Cromossômicas , Feminino , Feto , Humanos , Recém-Nascido , Cariotipagem , Gravidez , Diagnóstico Pré-NatalRESUMO
BACKGROUND: Non-invasive prenatal testing (NIPT) is extensively used in the detection of fetal trisomies 21, 18, and 13, which is promptly becoming a common clinical practice. Concerned about the clinical application of non-invasive detection of the fetal autosomal duplications or deletion. CASE PRESENTATION: A 34-year-old, healthy pregnant woman was referred to the First Affiliated Hospital of the Air Force Medical University. The ultrasound examination indicates that low-lying placenta, the fetus has a left ventricular bright spot and small amount of pericardial effusion. NIPT was chosen to further screen for fetal chromosomal abnormalities. NIPT results indicated an approximately 18 Mb deletion, which was verified by prenatal diagnosis. The chromosome microarray analysis (CMA) result showed about 19.2 Mb deletions in 21q11.2-q22.11. The karyotype analysis result showed 46,XN,del(21)(q11.2q22.1). Prenatal diagnosis was consistent with NIPT results, and the paternal karyotype revealed no obvious abnormalities. CONCLUSION: In this study, we successfully detected and diagnosed deletions of large fragments in chromosome 21 in a fetus using NIPT. This indicates that NIPT can provide effective genetic information for detecting fetal subchromosomal deletions/duplications.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 21/genética , Diagnóstico Pré-Natal/métodos , Adulto , Ácidos Nucleicos Livres/sangue , Ácidos Nucleicos Livres/genética , Feminino , Humanos , Cariotipagem , Análise de Sequência com Séries de Oligonucleotídeos , Gravidez , Análise de Sequência de DNA , Ultrassonografia Pré-NatalRESUMO
BACKGROUND: With the increasing availability of chromosomal microarray analysis (CMA) for congenital heart defect (CHD), genetic testing now faces new challenges due to results with uncertain clinical impact. Studies are needed to better define the penetrance of genetic variants. The aim of the study was to examine the association between CMA and CHDs in fetuses with normal karyotype. METHODS: This was a retrospective study of 190 fetuses with normal karyotype that underwent CMA after a diagnosis of CHD by fetal ultrasound. Invasive prenatal diagnosis was performed between January 2015 and December 2016 at the first affiliated hospital of Air Force Medical University. RESULTS: Chromosomal microarray analysis detected pathogenic copy number variants (pCNVs) in 13/190 (6.84%) fetuses, likely pCNVs in 5/190 (2.63%), and variants of unknown significance (VOUS) in 14/190 (7.37%). Among those with pCNVs, none (0%) yielded a normal live birth. Among those with likely pCNVs, 2/5 (40.0%) yielded a live birth. Among the fetuses with VOUS, 10/14 (71.5%) yielded a live birth. CONCLUSION: These results highlight the usefulness of CMA for prenatal genetic diagnosis of fetuses with CHDs and normal karyotype. In fetuses with CHD, the application of CMA could increase the detection rate of pCNVs causing CHDs. In this study, some VOUS were likely pathogenic, but additional studies are necessary to confirm these findings.
Assuntos
Variações do Número de Cópias de DNA/genética , Cardiopatias Congênitas/diagnóstico , Cardiopatias Congênitas/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Diagnóstico Pré-Natal/métodos , Adulto , Cromossomos/genética , Feminino , Testes Genéticos , Humanos , Cariótipo , Cariotipagem , Gravidez , Ultrassonografia Pré-Natal , Adulto JovemRESUMO
OBJECTIVE: To discuss the value of chromosomal microarray analysis (CMA) for the identification of DMD gene deletions during prenatal diagnosis. METHODS: G-banded karyotyping and CMA were performed on fetuses with ultrasonographic soft markers but no family history for Duchenne/Becker muscular dystrophy (DMD/BMD). Denaturing high-performance liquid chromatograghy (DHPLC) was used to detect DMD gene mutations in umbilical cord blood and peripheral blood samples from the mothers. RESULTS: For fetus 1, analysis of amniocytes showed a normal karyotype, while CMA detected a 119 kb deletion at Xp21.1 (32 565 489 - 32 681 461), which encompassed exons 10 to 16 of the DMD gene. The result was confirmed by DHPLC analysis. The mother was found to have loss of heterozygosity in the same region. For fetus 2, karyotyping of amniocytes also showed a normal male karyotype, while CMA detected a 254 kb deletion at Xp21.1 (32 104 604 - 32 358 874), which encompassed exons 41 to 44 of the DMD gene. The same deletion was not detected in the mother. DHPLC analysis confirmed the presence of both deletions. CONCLUSION: Two fetuses harboring DMD gene deletions but without a family history were discovered. CMA can improve the efficiency for detecting single gene diseases caused by deletions.
Assuntos
Distrofina/genética , Deleção de Genes , Achados Incidentais , Análise em Microsséries , Distrofia Muscular de Duchenne/genética , Éxons , Feminino , Feto , Humanos , Masculino , GravidezRESUMO
Bombyx mori and mulberry constitute a model of insect-host plant interactions. Urease hydrolyzes urea to ammonia and is important for the nitrogen metabolism of silkworms because ammonia is assimilated into silk protein. Silkworms do not synthesize urease and acquire it from mulberry leaves. We synthesized the artificial DNA sequence ureas using the codon bias of B. mori to encode the signal peptide and mulberry urease protein. A transgenic vector that overexpresses ure-as under control of the silkworm midgut-specific P2 promoter was constructed. Transgenic silkworms were created via embryo microinjection. RT-PCR results showed that urease was expressed during the larval stage and qPCR revealed the expression only in the midgut of transgenic lines. Urea concentration in the midgut and hemolymph of transgenic silkworms was significantly lower than in a nontransgenic line when silkworms were fed an artificial diet. Analysis of the daily body weight and food conversion efficiency of the fourth and fifth instar larvae and economic characteristics indicated no differences between transgenic silkworms and the nontransgenic line. These results suggested that overexpression of host plant urease promoted nitrogen metabolism in silkworms.
Assuntos
Bombyx/genética , Morus/parasitologia , Ureia/metabolismo , Urease/genética , Animais , Animais Geneticamente Modificados , Sequência de Bases , Bombyx/enzimologia , Bombyx/crescimento & desenvolvimento , DNA/química , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Vetores Genéticos , Larva , Masculino , Dados de Sequência Molecular , Morus/enzimologia , Folhas de Planta/genética , Regiões Promotoras Genéticas/genética , Ureia/análise , Urease/metabolismoRESUMO
The midgut is an important organ for digestion and absorption of nutrients and immune defense in the silkworm Bombyx mori. In an attempt to create a tool for midgut research, we cloned the 1080 bp P2 promoter sequence (P2P) of a highly expressed midgut-specific gene in the silkworm. The transgenic line (P2) was generated via embryo microinjection, in which the expression of EGFP was driven by P2P. There was strong green fluorescence only in the midgut of P2. RT-PCR and Western blot showed that P2P was a midgut-specific promoter with activity throughout the larval stage. A transgenic truncation experiment suggested that regions -305 to -214 and +107 to +181 were very important for P2P activity. The results of this study revealed that we have identified a midgut-specific promoter with a high level of activity in the silkworm that will aid future research and application of silkworm genes.
Assuntos
Bombyx/genética , Trato Gastrointestinal/citologia , Genes de Insetos , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Western Blotting , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Fluorescência , Engenharia Genética/métodos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Larva/genética , Larva/metabolismo , Microinjeções , Dados de Sequência Molecular , Organismos Geneticamente Modificados/genética , Organismos Geneticamente Modificados/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade da EspécieRESUMO
PURPOSE: The 12q terminal duplication is a chromosomal structural abnormality that has been rarely reported. The common clinical manifestations include intellectual disability and speech delay. We report two cases of patients with a duplication of chromosome 12q which was discovered incidentally during non-invasive prenatal genetic testing (NIPT). METHODS: Next generation sequencing-based NIPT and karyotype analysis confirmed the type and inheritance of the rearrangement, and chromosomal microarray-based analysis also confirmed the end replication. RESULTS: One patient had a 18Mb 12q24.21q24.33 duplication. The other patient had a12.04Mb12.q24.31q24.33 duplication and a 9.56Mb deletion in 18p11.32p11.22. The duplicated regions on chromosome 12 and the deletion on chromosome 18 in the patients were pathogenic, and the fetuses may have clinical characteristics, such as mental retardation, facial deformities, and psychomotor retardation. Ultimately, both pregnant women chose to terminate their pregnancy. CONCLUSION: The cases we reported show that NIPT cannot only detect conventional chromosomes, but can also detect microdeletions and microduplications, which broadens the scope of clinical application for NIPT and provides genetic information for high-risk pregnant women as early as possible.
Assuntos
Transtornos Cromossômicos , Cromossomos Humanos Par 12 , Feminino , Feto , Testes Genéticos , Humanos , Gravidez , Diagnóstico Pré-NatalRESUMO
BACKGROUND: Copy number variations (CNVs) involving the 17q12 region are associated with a broad range of clinical phenotypes. Deletion of the 17q12 chromosome results in structural or functional abnormalities in the kidney and urethra, type 5 diabetes (MODY5), and neurodevelopmental or neuropsychiatric disorders. Microduplication of 17q12 is rare and is associated with an increased risk of epilepsy and mental retardation. We studied the prenatal diagnosis of 17q12 microduplication and microdeletion syndrome in fetuses with congenital renal abnormalities. CASE PRESENTATION: We conducted a retrospective analysis of prenatal diagnoses in our hospital from January 2016 to April 2018. Abnormal renal ultrasound findings were present in 126 fetuses and the incidence of chromosomal abnormalities was 10.32%(13/126). Conventional karyotyping detected 7 of 126 fetuses as aneuploid (5.56%). In addition, chromosome microarray analysis (CMA) detected 6 fetuses(4.76%) with copy number variations (CNVs), of which 5 were shown to have 17q12 microdeletion syndrome and 1 had 17q12 microduplication syndrome. We followed up these pregnant women. The results of the testing had a significant impact on pregnancy outcome. The phenotypes of 17q12 microdeletions and microduplications vary widely, affecting patients in different ways, such as language delays, social deficiencies, and even abortion. CONCLUSIONS: The characteristics of 17q12 microdeletions and microduplications are so vague that the condition is often misdiagnosed or missed. This study demonstrated that karyotype analysis combined with CMA can significantly improve the diagnostic rate in prenatal diagnosis of CNVs, which can provide evidence for genetic counseling in such pregnancies.
RESUMO
OBJECTIVES: Cell-free fetal DNA has been widely used in prenatal genetic testing during recent years. We explored the feasibility of non-invasive prenatal testing (NIPT) for analysis of common fetal aneuploidies among pregnancies in northwest China. MATERIAL AND METHODS: A total of 8594 maternal blood samples were collected from October 2014 to December 2017 in the Department of Obstetrics and Gynecology at the First Affiliated Hospital of the Air Force Medical University. Cases with positive screening results by NIPT detection were validated using karyotype analysis. RESULTS: Of 8594 clinical pregnancies, 88 had positive NIPT results and 78 of 88 (88.6%) positive NIPT results were shown to be false-positive by amniotic fluid puncture and chromosome karyotyping analysis. There were 44 cases (49.44%) with trisomy 21, 18, and 13 syndromes (30 cases of trisomy 21, 9 cases of trisomy 18, and 5 cases of trisomy 13). There were 44 cases (50.56%) with sex chromosome abnormalities, including 11 cases with Turner syndrome (45, X), 17 cases with Triple X syndrome (47, XXX), 2 cases with Klinefelter syndrome (47, XXY), and 14 cases with 47, XYY syndrome (47, XYY). CONCLUSIONS: The accuracy, specificity, high efficiency, and acceptance of NIPT can effectively avoid birth defects and improve the quality of the birth population. We should deepen mining and analysis of the clinical data and explore ways to use NIPT. It is recommended that the NIPT guidelines be extended to low-risk patients to further explore the impact of a significant increase in screening.
Assuntos
Transtornos Cromossômicos/diagnóstico , Transtornos Cromossômicos/genética , Diagnóstico Pré-Natal/métodos , Ácidos Nucleicos Livres/sangue , Estudos de Viabilidade , Feminino , Humanos , Cariotipagem , GravidezRESUMO
Peptidoglycan recognition protein (PGRP) is an important pattern recognition receptor in innate immunity that is vital for bacterial recognition and defense in insects. Few studies report the role of PGRP in viral infection. Here we cloned two forms of PGRP from the model lepidopteran Bombyx mori: BmPGRP2-1 is a transmembrane protein, whereas BmPGRP2-2 is an intracellular protein. BmPGRP2-1 bound to diaminopimelic acid (DAP)-type peptidoglycan (PGN) to activate the canonical immune deficiency (Imd) pathway. BmPGRP2-2 knockdown reduced B. mori nucleopolyhedrovirus (BmNPV) multiplication and mortality in cell lines and in silkworm larvae, while its overexpression increased viral replication. Transcriptome and quantitative PCR (qPCR) results confirmed that BmPGRP2 negatively regulated phosphatase and tensin homolog (PTEN). BmPGRP2-2 expression was induced by BmNPV, and the protein suppressed PTEN-phosphoinositide 3-kinase (PI3K)/Akt signaling to inhibit cell apoptosis, suggesting that BmNPV modulates BmPGRP2-2-PTEN-PI3K/Akt signaling to evade host antiviral defense. These results demonstrate that the two forms of BmPGRP2 have different functions in host responses to bacteria and viruses.
Assuntos
Bombyx/imunologia , Bombyx/metabolismo , Proteínas de Transporte/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Proteínas de Insetos/metabolismo , Animais , Bactérias/imunologia , Bombyx/microbiologia , Bombyx/virologia , Proteínas de Transporte/genética , Linhagem Celular , Clonagem Molecular , Expressão Gênica , Proteínas de Insetos/genética , Modelos Biológicos , Peptidoglicano/imunologia , Peptidoglicano/metabolismo , Transdução de Sinais , Vírus/imunologiaRESUMO
Introns are important for regulating gene expression. BmAPN4, which has a 5'-UTR upstream intron (5 UI), is specifically expressed in the entire silkworm midgut. In our previous study, the promoter region upstream of the 5 UI of BmAPN4 was cloned and identified as the P3 promoter (P3P) with activity only in the anterior midgut. In this study, the sequence consisting of the P3P and the 5 UI was cloned and named as P3P+5 UI. A transgenic vector was constructed in which EGFP was controlled by P3P+5 UI. Transgenic P3+5 UI silkworms were generated by embryo microinjection. RT-PCR showed P3P+5 UI activity throughout the larval stage. Intense green fluorescence was seen only in the entire midgut of P3+5 UI silkworms and expression was confirmed by RT-PCR. qPCR revealed that expression of EGFP in the anterior midgut of P3+5 UI silkworms was 64% higher than in P3 silkworms, indicating the 5 UI sustained intron-mediated enhancement of gene expression. These results suggested that the BmAPN4 5 UI affected the level and site of expression. The 5 UI was cloned and added behind P2P, another specific promoter with activity only in the anterior midgut of silkworm, to construct the P2P+5 UI and transgenic P2+5 UI silkworms. Expression patterns were the same for P2P+5 UI and P2P, suggesting that the 5UI of BmAPN4 did not affect P2P. This study found that the BmAPN4 5 UI affected the amount and location of gene expression. Its influence appeared to be dependent on a specific promoter.
Assuntos
Regiões 5' não Traduzidas/genética , Bombyx/genética , Antígenos CD13/genética , Genes de Insetos , Regiões Promotoras Genéticas , Animais , Animais Geneticamente Modificados , Bombyx/metabolismo , Antígenos CD13/metabolismo , Expressão Gênica , Vetores Genéticos , Íntrons , Larva/genética , Larva/metabolismoRESUMO
The piggyBac transposon is the most widely used vector for generating transgenic silkworms. The silkworm genome contains multiple piggyBac-like sequences that might influence the genetic stability of transgenic lines. To investigate the postintegration stability of piggyBac in silkworms, we used random insertion of the piggyBac [3 × p3 EGFP afm] vector to generate a W chromosome-linked transgenic silkworm, named W-T. Results of Southern blot and inverse PCR revealed the insertion of a single copy in the W chromosome of W-T at a standard TTAA insertion site. Investigation of 11 successive generations showed that all W-T females were EGFP positive and all males were EGFP negative; PCR revealed that the insertion site was unchanged in W-T offspring. These results suggested that endogenous piggyBac-like elements did not affect the stability of piggyBac inserted into the silkworm genome.
Assuntos
Animais Geneticamente Modificados/genética , Bombyx/genética , Elementos de DNA Transponíveis/genética , Animais , Sequência de Bases , Feminino , Proteínas de Fluorescência Verde/genética , Masculino , TransgenesRESUMO
Bombyx mori nucleopolyhedrovirus (BmNPV) is the primary pathogen of silkworms, causing severe economic losses in sericulture. To create antiviral silkworm strains, we constructed a transgenic vector in which the dsRNA for five tandem BmNPV genes was controlled by the BmNPV hr3 enhancer and IE1 promoter. The antivirus gene Bmlipase-1 was driven by B. mori midgut-specific promoter P2. Transgenic strains (SW-H) were generated via embryo microinjection using the practical silkworm strain SW. After infection with a high dose of BmNPV, the survival rates of SW-H and non-transgenic SW were 64% and 13%, respectively. SW-H could be the first transgenic animal that is highly antiviral and that might inhibit the virus at multiple stages of infection.