RESUMEN
Gene-knockout pigs have important applications in agriculture and medicine. Compared with CRISPR/Cas9, Adenine base editor (ABE) convert single A·T pairs to G·C pairs in the genome without generating DNA double-strand breaks, and this method has higher accuracy and biosafety in pig genetic modification. However, the application of ABE in pig gene knockout is limited by protospacer-adjacent motif sequences and the base-editing window. Alternative mRNA splicing is an important mechanism underlying the formation of proteins with diverse functions in eukaryotes. Spliceosome recognizes the conservative sequences of splice donors and acceptors in a precursor mRNA. Mutations in these conservative sequences induce exon skipping, leading to proteins with novel functions or to gene inactivation due to frameshift mutations. In this study, adenine base-editing-mediated exon skipping was used to expand the application of ABE in the generation of gene knockout pigs. We first constructed a modified "all-in-one" ABE vector suitable for porcine somatic cell transfection that contained an ABE for single-base editing and an sgRNA expression cassette. The "all-in-one" ABE vector induced efficient sgRNA-dependent A-to-G conversions in porcine cells during single base-editing of multiple endogenous gene loci. Subsequently, an ABE system was designed for single adenine editing of the conservative splice acceptor site (AG sequence at the 3' end of the intron 5) and splice donor site (GT sequence at the 5' end of the intron 6) in the porcine gene GHR; this method achieved highly efficient A-to-G conversion at the cellular level. Then, porcine single-cell colonies carrying a biallelic A-to-G conversion in the splice acceptor site in the intron 5 of GHR were generated. RT-PCR indicated exon 6 skipped at the mRNA level. Western blotting revealed GHR protein loss, and gene sequencing showed no sgRNA-dependent off-target effects. These results demonstrate accurate adenine base-editing-mediated exon skipping and gene knockout in porcine cells. This is the first proof-of-concept study of adenine base-editing-mediated exon skipping for gene regulation in pigs, and this work provides a new strategy for accurate and safe genetic modification of pigs for agricultural and medical applications.
Asunto(s)
Adenina , Edición Génica , Adenina/metabolismo , Animales , Sistemas CRISPR-Cas/genética , Línea Celular , Exones/genética , Edición Génica/métodos , Técnicas de Inactivación de Genes , PorcinosRESUMEN
Bama minipig is a unique miniature swine bred from China. Their favorable characteristics include delicious meat, strong adaptability, tolerance to rough feed, and high levels of stress tolerance. Unfavorable characteristics are their low lean meat percentage, high fat content, slow growth rate, and low feed conversion ratio. Genome-editing technology using CRISPR/Cas9 efficiently knocked out the myostatin gene (MSTN) that has a negative regulatory effect on muscle production, effectively promoting pig muscle growth and increasing lean meat percentage of the pigs. However, CRISPR/Cas9 genome editing technology is based on random mutations implemented by DNA double-strand breaks, which may trigger genomic off-target effects and chromosomal rearrangements. The application of CRISPR/Cas9 to improve economic traits in pigs has raised biosafety concerns. Base editor (BE) developed based on CRISPR/Cas9 such as cytosine base editor (CBE) effectively achieve targeted modification of a single base without relying on DNA double-strand breaks. Hence, the method has greater safety in the genetic improvement of pigs. The aim of the present study is to utilize a modified CBE to generate MSTN-knockout cells of Bama minipigs. Our results showed that the constructed "all-in-one"-modified CBE plasmid achieved directional conversion of a single C·G base pair to a T·A base pair of the MSTN target in Bama miniature pig fibroblast cells. We successfully constructed multiple single-cell colonies of Bama minipigs fibroblast cells carrying the MSTN premature termination and verified that there were no genomic off-target effects detected. This study provides a foundation for further application of somatic cell cloning to construct MSTN-edited Bama minipigs that carry only a single-base mutation and avoids biosafety risks to a large extent, thereby providing experience and a reference for the base editing of other genetic loci in Bama minipigs.
Asunto(s)
Citosina/metabolismo , Fibroblastos/citología , Edición Génica/métodos , Miostatina/genética , Animales , Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas , Células Cultivadas , Codón de Terminación , Fibroblastos/metabolismo , Plásmidos/genética , Porcinos , Porcinos Enanos , TransfecciónRESUMEN
OBJECTIVES: Guangdong Small-ear Spotted (GDSS) pigs are a pig breed native to China that possesses unfortunate disadvantages, such as slow growth rate, low lean-meat percentage, and reduced feed utilization. In contrast to traditional genetic breeding methods with long cycle time and high cost, CRISPR/Cas9-mediated gene editing for the modification of the pig genome can quickly improve production traits, and therefore this technique exhibits important potential in the genetic improvement and resource development of GDSS pigs. In the present study, we aimed to establish an efficient CRISPR/Cas9-mediated gene-editing system for GDSS pig cells by optimizing the electrotransfection parameters, and to realize efficient CRISPR/Cas9-mediated gene editing of GDSS pig cells. RESULTS: After optimization of electrotransfection parameters for the transfection of GDSS pig cells, we demonstrated that a voltage of 150 V and a single pulse with a pulse duration of 20 ms were the optimal electrotransfection parameters for gene editing in these cells. In addition, our study generated GDSS pig single-cell colonies with biallelic mutations in the myostatin (MSTN) gene and insulin-like growth factor 2 (IGF2) intron-3 locus, which play an important role in pig muscle growth and muscle development. The single-cell colonies showed no foreign gene integration or off-target effects, and maintained normal cell morphology and viability. These gene-edited, single-cell colonies can in the future be used as donor cells to generate MSTN- and IGF2-edited GDSS pigs using somatic cell nuclear transfer (SCNT). CONCLUSIONS: This study establishes the foundation for genetic improvement and resource development of GDSS pigs using CRISPR/Cas9-mediated gene editing combined with SCNT.
Asunto(s)
Edición Génica/métodos , Factor II del Crecimiento Similar a la Insulina/genética , Miostatina/genética , Transfección/métodos , Animales , Sistemas CRISPR-Cas , Línea Celular , Fenómenos Electromagnéticos , Mutación , Selección Artificial , Análisis de la Célula Individual , PorcinosRESUMEN
CRISPR/Cas9-mediated genome editing technology is a simple and highly efficient and specific genome modification approach with wide applications in the animal industry. CRISPR/Cas9-mediated genome editing combined with somatic cell nuclear transfer rapidly constructs gene-edited somatic cell-cloned pigs for the genetic improvement of traits or simulation of human diseases. Chinese Bama pigs are an excellent indigenous minipig breed from Bama County of China. Research on genome editing of Chinese Bama pigs is of great significance in protecting its genetic resource, improving genetic traits and in creating disease models. This study aimed to address the disadvantages of slow growth and low percentage of lean meat in Chinese Bama pigs and to knock out the myostatin gene (MSTN) by genome editing to promote growth and increase lean meat production. We first used CRISPR/Cas9-mediated genome editing to conduct biallelic knockout of the MSTN, followed by somatic cell nuclear transfer to successfully generate MSTN biallelic knockout Chinese Bama pigs, which was confirmed to have significantly faster growth rate and showed myofibre hyperplasia when they reached sexual maturity. This study lays the foundation for the rapid improvement of production traits of Chinese Bama pigs and the generation of gene-edited disease models in this breed.
Asunto(s)
Sistemas CRISPR-Cas , Miostatina/genética , Porcinos Enanos/genética , Animales , Femenino , Técnicas de Inactivación de Genes/veterinaria , Masculino , Fibras Musculares Esqueléticas/fisiología , Técnicas de Transferencia Nuclear/veterinaria , Carne de Cerdo , Porcinos , Porcinos Enanos/crecimiento & desarrolloRESUMEN
In mammals, leptin is a peripheral satiety factor that inhibits feeding by regulating a variety of appetite-related hormones in the brain. However, most of the previous studies examining leptin in fish feeding were performed with mammalian leptins, which share very low sequence homologies with fish leptins. To elucidate the function and mechanism of endogenous fish leptins in feeding regulation, recombinant goldfish leptin-AI and leptin-AII were expressed in methylotrophic yeast and purified by immobilized metal ion affinity chromatography (IMAC). By intraperitoneal (IP) injection, both leptin-AI and leptin-AII were shown to inhibit the feeding behavior and to reduce the food consumption of goldfish in 2 h. In addition, co-treatment of leptin-AI or leptin-AII could block the feeding behavior and reduce the food consumption induced by neuropeptide Y (NPY) injection. High levels of leptin receptor (lepR) mRNA were detected in the hypothalamus, telencephalon, optic tectum and cerebellum of the goldfish brain. The appetite inhibitory effects of leptins were mediated by downregulating the mRNA levels of orexigenic NPY, agouti-related peptide (AgRP) and orexin and upregulating the mRNA levels of anorexigenic cocaine-amphetamine-regulated transcript (CART), cholecystokinin (CCK), melanin-concentrating hormone (MCH) and proopiomelanocortin (POMC) in different areas of the goldfish brain. Our study, as a whole, provides new insights into the functions and mechanisms of leptins in appetite control in a fish model.
Asunto(s)
Ingestión de Alimentos/efectos de los fármacos , Carpa Dorada/fisiología , Leptina/administración & dosificación , Leptina/genética , Animales , Encéfalo/metabolismo , Conducta Alimentaria/efectos de los fármacos , Proteínas de Peces/administración & dosificación , Proteínas de Peces/genética , Proteínas de Peces/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Leptina/farmacología , Neuropéptido Y/administración & dosificación , Receptores de Leptina/genética , Receptores de Leptina/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
Alzheimer's disease (AD) is an age-related progressive neurodegenerative disorder that leads to cognitive impairment and memory loss. Emerging evidence suggests that autophagy plays an important role in the pathogenesis of AD through the regulation of amyloid-beta (Aß) and tau metabolism, and that autophagy dysfunction exacerbates amyloidosis and tau pathology. Therefore, targeting autophagy may be an effective approach for the treatment of AD. Animal models are considered useful tools for investigating the pathogenic mechanisms and therapeutic strategies of diseases. This review aims to summarize the pathological alterations in autophagy in representative AD animal models and to present recent studies on newly discovered autophagy-stimulating interventions in animal AD models. Finally, the opportunities, difficulties, and future directions of autophagy targeting in AD therapy are discussed.
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Enfermedad de Alzheimer , Animales , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/veterinaria , Péptidos beta-Amiloides , Autofagia/fisiología , Modelos AnimalesRESUMEN
Gene-knockout pigs have important applications in agriculture and medicine. Compared with CRISPR/Cas9 and cytosine base editing (CBE) technologies, adenine base editing (ABE) shows better safety and accuracy in gene modification. However, because of the characteristics of gene sequences, the ABE system cannot be widely used in gene knockout. Alternative splicing of mRNA is an important biological mechanism in eukaryotes for the formation of proteins with different functional activities. The splicing apparatus recognizes conserved sequences of the 5' end splice donor and 3' end splice acceptor motifs of introns in pre-mRNA that can trigger exon skipping, leading to the production of new functional proteins, or causing gene inactivation through frameshift mutations. This study aimed to construct a MSTN knockout pig by inducing exon skipping with the aid of the ABE system to expand the application of the ABE system for the preparation of knockout pigs. In this study, first, we constructed ABEmaxAW and ABE8eV106W plasmid vectors and found that their editing efficiencies at the targets were at least sixfold and even 260-fold higher than that of ABEmaxAW by contrasting the editing efficiencies at the gene targets of endogenous CD163, IGF2, and MSTN in pigs. Subsequently, we used the ABE8eV106W system to realize adenine base (the base of the antisense strand is thymine) editing of the conserved splice donor sequence (5'-GT) of intron 2 of the porcine MSTN gene. A porcine single-cell clone carrying a homozygous mutation (5'-GC) in the conserved sequence (5'-GT) of the intron 2 splice donor of the MSTN gene was successfully generated after drug selection. Unfortunately, the MSTN gene was not expressed and, therefore, could not be characterized at this level. No detectable genomic off-target edits were identified by Sanger sequencing. In this study, we verified that the ABE8eV106W vector had higher editing efficiency and could expand the editing scope of ABE. Additionally, we successfully achieved the precise modification of the alternative splice acceptor of intron 2 of the porcine MSTN gene, which may provide a new strategy for gene knockout in pigs.
Asunto(s)
Adenina , Edición Génica , Animales , Porcinos , Exones/genética , Mutación , Técnicas de Inactivación de GenesRESUMEN
Different kinds of orbitozygomatic fractures lead to different levels of orbital structural destructions. Although the magnitude of the management of orbitozygomatic fractures varies considerably, an unsolved and important question remains regarding how to select the best surgical modality according to different kinds of fractures. Among 26 cases of unilateral noncomminuted orbitozygomatic fractures, a segmental osteotomy technique was used to repair the displaced orbitozygomatic complex. The fragment contributing to the orbital structure was mobilized and reduced, whereas the fragment without contribution to the orbital structure was not mobilized and left in situ. Next, the cuneate fragment was imbricated with the orbital osteotomy sites unilaterally or bilaterally. Miniplates and screws were used to span the osteotomy sites and provided fixation once the alignment of the orbit was achieved. The mean difference in volume between fractured orbits preoperatively and postoperatively was 2.47 mL. The mean difference in ocular globe projection was 2.76 mm. When postoperative results were compared with those of the preoperative site, statistically significant difference was noted. At follow-up 1 year postoperatively, the management of orbitozygomatic fractures using segmental osteotomy reduces complications and attains aesthetically satisfying results. Subjective assessment of the patients' globe position found that 88.5% of the patients were satisfied with the outcome, and 11.5% of the patients found it unacceptable. Compared with the traditional method, segmental osteotomy is a simple technique requiring less dissection and can reconstruct the orbital anatomic structure and restore globe position effectively.
Asunto(s)
Fracturas Orbitales/cirugía , Osteotomía/métodos , Fracturas Cigomáticas/cirugía , Adolescente , Adulto , Placas Óseas , Tornillos Óseos , Niño , Estética , Femenino , Estudios de Seguimiento , Humanos , Imagenología Tridimensional , Luxaciones Articulares/cirugía , Masculino , Órbita/cirugía , Osteotomía/instrumentación , Planificación de Atención al Paciente , Satisfacción del Paciente , Procedimientos de Cirugía Plástica/instrumentación , Procedimientos de Cirugía Plástica/métodos , Tomografía Computarizada Espiral , Resultado del Tratamiento , Adulto Joven , Cigoma/cirugíaRESUMEN
OBJECTIVE: To explore the toxicological mechanism of hydroquinone in human bronchial epithelial cells and to investigate whether DNA polymerase beta is involved in protecting cells from damage caused by hydroquinone. METHODS: DNA polymerase beta knock-down cell line was established via RNA interference as an experimental group. Normal human bronchial epithelial cells and cells transfected with the empty vector of pEGFP-C1 were used as controls. Cells were treated with different concentrations of hydroquinone (ranged from 10 micromol/L to 120 micromol/L) for 4 hours. MTT assay and Comet assay [single-cell gel electrophoresis (SCGE)] were performed respectively to detect the toxicity of hydroquinone. RESULTS: MTT assay showed that DNA polymerase beta knock-down cells treated with different concentrations of hydroquinone had a lower absorbance value at 490 nm than the control cells in a dose-dependant manner. Comet assay revealed that different concentrations of hydroquinone caused more severe DNA damage in DNA polymerase beta knock-down cell line than in control cells and there was no significant difference in the two control groups. CONCLUSIONS: Hydroquinone has significant toxicity to human bronchial epithelial cells and causes DNA damage. DNA polymerase beta knock-down cell line appears more sensitive to hydroquinone than the control cells. The results suggest that DNA polymerase beta is involved in protecting cells from damage caused by hydroquinone.
Asunto(s)
Citotoxinas/toxicidad , Daño del ADN , ADN Polimerasa beta/fisiología , Hidroquinonas/toxicidad , Bronquios/citología , Bronquios/efectos de los fármacos , Células Cultivadas , Ensayo Cometa , ADN Polimerasa beta/antagonistas & inhibidores , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Humanos , Interferencia de ARNRESUMEN
Leptin is a peripheral satiety hormone that also plays important roles in energy homeostasis in vertebrates ranging from fish to mammals. In teleost fish, however, the regulatory mechanism for leptin gene expression still remains unclear. In this study, we found that glucagon, a key hormone in glucose homeostasis, was effective at elevating the leptin-AI and leptin-AII transcript levels in goldfish liver via both in vivo intraperitoneal injection and in vitro cells incubation approaches. The responses of leptin-AI and leptin-AII mRNA to glucagon treatment were highly comparable. In contrast, blockade of local glucagon action could reduce the basal and induced leptin-AI and leptin-AII mRNA expression. The stimulation of leptin levels by glucagon was caused by the activation of adenylate cyclase (AC)/cyclic-AMP (cAMP)/ protein kinase A (PKA), and probably cAMP response element-binding protein (CREB) cascades. Our study described the effect and signal transduction mechanism of glucagon on leptin gene expression in goldfish liver, and may also provide new insight into leptin as a mediator in the regulatory network of energy metabolism in the fish model.
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Glucagón/farmacología , Carpa Dorada/metabolismo , Leptina/metabolismo , Hígado/metabolismo , Adenilil Ciclasas/metabolismo , Animales , AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Carpa Dorada/genética , Leptina/genética , Transducción de Señal/efectos de los fármacosRESUMEN
A sequence-specific M1GS ribozyme (M1-T3) was constructed by covalently linking an oligonucleotide (guide sequence,GS) to the 3' terminus of M1 RNA ,the catalytic subunit of RNase P from Escherichia coli. The engineered ribozyme is targeted to the mRNA sequence encoding a protein kinase (UL97) of HCMV and could effectively cleave the mRNA segment in vitro. Further studies about the significance of some structural elements in the M1 GS (e.g. the 3' CCA tail sequence and a bridge sequence between the 3' terminus of M1 RNA and the 5' terminus of the GS) were carried out. The results showed that the bridge sequence of 88 nucleotides in a mutated M1 GS (i.e. M1-T3*) dramatically increased the cleavage activity to the substrate in vitro. Moreover, the 3'CCA tail sequence was confirmed to be a necessary element for the cleavage activity of M1 GS ribozyme. These data we got in the study will help in understanding the interaction between the M1 GS RNA and its substrate,and will markedly facilitate the research of a general gene targeting agent for anti-HCMV applications.
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Regiones no Traducidas 5'/metabolismo , Proteínas de Escherichia coli/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , ARN Catalítico/metabolismo , Ribonucleasa P/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/síntesis química , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , ARN Viral/metabolismo , Ribonucleasa P/síntesis química , ARN Pequeño no TraducidoRESUMEN
To clone novel myelin protein related genes, two human ESTs, which shared significant similarity with the human myelin protein zero gene, were found by the comparison of homologue between the cDNA coding region sequences of MPZ gene and the EST database of NCBI. An 801 bp EST contig was assembled, which was 100% identical with a 128 kb genomic sequence, mapped to 1q24. A 435 bp open reading frame (ORF) within the 801 bp contig was shown by computer analysis. Two primers designed according to the sequence of the contig, were coupled with the primers(lambdagt10-5 and gt10-5) on the sequences flanking cloning site of the cDNA library vector to amplify the cDNA library sequences by nested PCR. New primers, designed based on novel cDNA sequences, were used for the PCR amplification with lambdagt10-5 and gt10-5 in the same way as above. Finally, the human myelin protein zero like gene isoform I and II (MPZL1a, MPZL1b GenBank AF095727, AF092424) were cloned. Comparison of gene and protein structures between MPZL1 and MPZ revealed that MPZL1 is the second member of MPZ family. Mutation analysis of MPZL1 gene was performed in 24 Charcot-Marie-Tooth disease (CMT) families and 26 nonsyndrome deafness families, but no mutation was found.
RESUMEN
OBJECTIVE: To isolate the wild-type virulent phage of Helicobacter pylori (Hp) and simulate the treatments in vitro to investigate the methods for oral Hp-assisted penetration of the phage through the gastric barrier and offspring phage release for infection and treatment of gastrointestinal Hp. METHODS: The Hp strain was cultured with the candle cylinder method and the virulent phage was isolated by single plate or double plate experiment. A simulated gastric juice was applied and the bactericidal effect of the phage was tested with double flats experiment. RESULTS: After a 1.5-h treatment in simulated gastric juice, the orally derived Hp-borne phage was still capable of forming plaques while the control phage was not. CONCLUSION: The oral Hp can help the phage resist the gastric juice and then infect the gastrointestinal Hp.
Asunto(s)
Bacteriófagos/aislamiento & purificación , Bacteriófagos/fisiología , Tracto Gastrointestinal/microbiología , Infecciones por Helicobacter/terapia , Helicobacter pylori/virología , Humanos , VirulenciaRESUMEN
Endophytic fungi are little known for their role in gibberellins (GAs) synthesis and abiotic stress resistance in crop plants. We isolated 10 endophytes from the roots of field-grown soybean and screened their culture filtrates (CF) on the GAs biosynthesis mutant rice line - Waito-C. CF bioassay showed that endophyte GMH-1B significantly promoted the growth of Waito-C compared with controls. GMH-1B was identified as Penicillium minioluteum LHL09 on the basis of ITS regions rDNA sequence homology and phylogenetic analyses. GC/MS-SIM analysis of CF of P. minioluteum revealed the presence of bioactive GA(4) and GA(7). In endophyte-soybean plant interaction, P. minioluteum association significantly promoted growth characteristics (shoot length, shoot fresh and dry biomasses, chlorophyll content, and leaf area) and nitrogen assimilation, with and without sodium chloride (NaCl)-induced salinity (70 and 140 mM) stress, as compared with control. Field-emission scanning electron microcopy showed active colonization of endophyte with host plants before and after stress treatments. In response to salinity stress, low endogenous abscisic acid and high salicylic acid accumulation in endophyte-associated plants elucidated the stress mitigation by P. minioluteum. The endophytic fungal symbiosis of P. minioluteum also increased the daidzein and genistein contents in the soybean as compared with control plants, under salt stress. Thus, P. minioluteum ameliorated the adverse effects of abiotic salinity stress and rescued soybean plant growth by influencing biosynthesis of the plant's hormones and flavonoids.
Asunto(s)
Endófitos/fisiología , Glycine max/fisiología , Penicillium/fisiología , Endófitos/genética , Endófitos/aislamiento & purificación , Datos de Secuencia Molecular , Oryza/crecimiento & desarrollo , Oryza/microbiología , Penicillium/genética , Penicillium/aislamiento & purificación , Raíces de Plantas/química , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/microbiología , Raíces de Plantas/fisiología , Salinidad , Cloruro de Sodio/metabolismo , Glycine max/química , Glycine max/crecimiento & desarrollo , Glycine max/microbiología , Estrés FisiológicoRESUMEN
Based on the sequence of BAC (Bacterial Artificial Chromosome) along with the Cre/lox P system, the gene-targeting vectors to multiple loci of the repetitive internal transcribed spacers between rDNA genes in Leghorn chicken were constructed. The key material of multiple loci gene targeting in vivo would be obtained. First, the plasmid of pYLSV-TDN with TK, HRDS2, and Neo genes was constructed. The TK-HRDS2-Neo DNA fragment obtained from the plasmid of pYLSV-TDN was digested by Not I/HindIII and inserted into the upstream of the lox P site of BAC plasmid for obtaining the selective vector of BAC-TDN. The expression vector of pYLVS-GID with EGFP, hIFN genes, and HRDS1 was then obtained. The plasmid of BAC-TDN-VS-GID was obtained by cotransformation of the selective vector of BAC-TDN and the expression vector of pYLVS-GID to E. coli NS3529 through the action of Cre/lox P system. The gene-targeting vector of BAC-TDN-GID to multiple loci of the ITS region in Leghorn chicken was obtained by cleaving the sequence of pYLVS with the homing endonuclease of I -Sce I and ligating with the linker of LS. The insertion and the insert direction of DNA fragments were identified by restriction digestion or PCR and sequencing in each clone. The significance of the technique ofgene-targeting vector to multiple loci are shown as follows. First, the targeting loci were increased to 100 - 300. Second, the problems of unstable expression of inserted genes were partially solved. Third, the need for safety against toxicity integration was resolved. Fourth, the forbidden zone of gene integrating on the repetitive DNA sequences was broken through.
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Cromosomas Artificiales Bacterianos/genética , ADN Espaciador Ribosómico/genética , Vectores Genéticos/genética , Integrasas/genética , Animales , Sitios de Ligazón Microbiológica/genética , Clonación Molecular , ADN/genética , ADN/metabolismo , Enzimas de Restricción del ADN/metabolismo , Escherichia coli/genética , Proteínas Fluorescentes Verdes/genética , Humanos , Interferón gamma/genética , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/genética , Transformación GenéticaRESUMEN
Mouse Pem, a homeobox gene, encodes a protein consisting of 210 amino acid residues. To study the function of mouse Pem protein, we used the yeast two-hybrid system to screen the library of 7-day mouse embryo with full-length mouse Pem cDNA. Fifty-two colonies were obtained after 1.57 x 10(8) colonies were screened by nutrition limitation and beta-galactosidase assay. Seven individual insert fragments were obtained from the library, and three of them were identified, one of which was confirmed to be the cell division cycle 37 (Cdc37) homolog gene by sequencing. The interaction between mouse Pem and Cdc37 homolog was then confirmed by glutathione S-transferase pull-down assay, and the possible interaction model was suggested.
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Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Homeodominio/química , Proteínas de Homeodominio/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Ratones , Datos de Secuencia Molecular , Unión Proteica , Técnicas del Sistema de Dos HíbridosRESUMEN
Seven sequence-specific ribozymes (M1GS RNAs) derived in vitro from the catalytic RNA subunit of Escherichia coli RNase P and targeting the mRNAs transcribed by the UL54 gene encoding the DNA polymerase of human cytomegalovirus were screened from 11 ribozymes that were designed based on four rules: (1) the NCCA-3' terminal must be unpaired with the substrate; (2) the guide sequence (GS) must be at least 12 nt in length; (3) the eighth nucleotide must be U, counting from the site -1; and (4) around the cleavage site, the sites -1/+1/+2 must be U/G/C or C/G/C. Further investigation of the factors affecting the cleavage effect and the optimal ratio for M1GS/substrate was carried out. It was determined that the optimal ratio for M1GS/substrate was 2:1 and too much M1GS led to substrate degrading. As indicated above, several M1GS that cleaved HCMV UL54 RNA segments in vitro were successfully designed and constructed. Our studies support the use of ribozyme M1GS as antisense molecules to silence HCMV mRNA in vitro, and using the selection procedure as a general approach for the engineering of RNase P ribozymes.