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1.
Transgenic Res ; 29(1): 149-163, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31927726

RESUMO

Myostatin (MSTN), a member of the transforming growth factor-ß superfamily, is a negative regulator of muscle growth and development. Disruption of the MSTN gene in various mammalian species markedly promotes muscle growth. Previous studies have mainly focused on the disruption of the MSTN peptide coding region in pigs but not on the modification of the signal peptide region. In this study, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) system was used to successfully introduce two mutations (PVD20H and GP19del) in the MSTN signal peptide region of the indigenous Chinese pig breed, Liang Guang Small Spotted pig. Both mutations in signal peptide increased the muscle mass without inhibiting the production of mature MSTN peptide in the cells. Histological analysis revealed that the enhanced muscle mass in MSTN+/PVD20H pig was mainly due to an increase in the number of muscle fibers. The expression of MSTN in the longissimus dorsi muscle of MSTN+/PVD20H and MSTNKO/PVD20H pigs was significantly downregulated, whereas that of myogenic regulatory factors, including MyoD, Myogenin, and Myf-5, was significantly upregulated when compared to those in the longissimus dorsi muscle of wild-type pigs. Meanwhile, the mutations also activated the PI3K/Akt pathway. The results of this study indicated that precise editing of the MSTN signal peptide can enhance porcine muscle development without markedly affecting the expression of mature MSTN peptide, which could exert other beneficial biological functions in the edited pigs.

2.
Front Cell Dev Biol ; 7: 286, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31803742

RESUMO

Bone morphogenetic protein 15 (BMP15) is strongly associated with animal reproduction and woman reproductive disease. As a multifunctional oocyte-specific secret factor, BMP15 controls female fertility and follicular development in both species-specific and dosage-sensitive manners. Previous studies found that BMP15 played a critical role in follicular development and ovulation rate in mono-ovulatory mammalian species, especially in sheep and human, but study on knockout mouse model implied that BMP15 possibly has minimal impact on female fertility of poly-ovulatory species. However, this needs to be validated in other poly-ovulatory species. To investigate the regulatory role of BMP15 on porcine female fertility, we generated a BMP15-knockdown pig model through somatic nuclear transfer technology. The BMP15-knockdown gilts showed markedly reduced fertility accompanied by phenotype of dysplastic ovaries containing significantly declined number of follicles, increased number of abnormal follicles, and abnormally enlarged antral follicles resulting in disordered ovulation, which is remarkably different from the unchanged fertility observed in BMP15 knockout mice. Molecular and transcriptome analysis revealed that the knockdown of BMP15 significantly affected both granulosa cells (GCs) and oocytes development, including suppression of cell proliferation, differentiation, and follicle stimulating hormone receptor (Fshr) expression, leading to premature luteinization and reduced estradiol (E2) production in GCs, and simultaneously decreased quality and meiotic maturation of oocyte. Our results provide in vivo evidence of the essential role of BMP15 in porcine ovarian and follicular development, and new insight into the complicated regulatory function of BMP15 in female fertility of poly-ovulatory species.

3.
Yi Chuan ; 41(10): 939-949, 2019 Oct 20.
Artigo em Chinês | MEDLINE | ID: mdl-31624056

RESUMO

Mutations in Hypoxanthine-guanine Phosphoribosyltransferase1 (HPRT1) gene can lead to metabolic disorder of hypoxanthine and guanine metabolism, and other severe symptoms such as hypophrenia, gout, and kidney stones, called the Lesch-Nyhan disease (LND). Although the mutations are widely distributed throughout the HPRT1 gene, there are some isolated hot spots. In this study, we aim to introduce two previously reported hot spots, c.508 C>T and c.151 C>T, which could lead to premature translational termination in HPRT1 gene. Through CRISPR/Cas9 mediated homology-directed repair (HDR) by using single-stranded oligo-deoxyribonucleotides (ssODN) as donor template, we obtained cell clones containing these two mutations in HEK293T or HeLa cells. Targeted mutation of c.508 C>T and c.151 C>T reached to 16.3% and 10%, respectively. We further detect HPRT1 protein levels with Western blot and enzyme activity with 6-TG in 5 different cell clones. HPRT1 protein and its enzymatic activity both was hardly detected in homozygous mutant cells, while reduced HPRT1 protein expression and enzymatic activity was detected in heterozygous mutant cells. Our study will be beneficial to those who working on generation of cell or animal models of HRPT1 mutations, and provides a basis for further investigations on the genetic mechanism of Lesch-Nyhan disease.


Assuntos
Sistemas CRISPR-Cas , Hipoxantina Fosforribosiltransferase/genética , Mutação Puntual , Células HEK293 , Células HeLa , Humanos , Síndrome de Lesch-Nyhan/genética
4.
Front Immunol ; 10: 1846, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31440241

RESUMO

Porcine reproductive and respiratory syndrome virus (PRRSV) 1 and 2 differ in their recognition of CD163. Substitution of porcine CD163 SRCR5 domain with a human CD163-like SRCR8 confers resistance to PRRSV 1 but not PRRSV 2. The deletion of CD163 SRCR5 has been shown to confer resistance to PRRSV 1 in vivo and both PRRSV 1 and 2 in vitro. However, the anti-PRRSV 2 activity of modifying the CD163 SRCR5 domain has not yet been reported. Here, we describe the highly efficient generation of two pig breeds (Liang Guang Small Spotted and Large White pigs) lacking a short region of CD163 SRCR5, including the ligand-binding pocket. We generated a large number of gene-edited Large White pigs of the F0 generation for use in viral challenge studies. The results of this study show that these pigs are completely resistant to infection by species 2 PRRSV, JXA1, and MY strains. There were no clinical symptoms, pathological abnormalities, viremia, or anti-PRRSV antibodies in the CD163 SRCR5-edited pigs compared to wild-type controls after viral challenge. Porcine alveolar macrophages (PAMs) isolated from CD163 SRCR5-edited Large White pigs also displayed resistance to PRRSV in vitro. In addition, CD163 SRCR5-edited PAMs still exhibited a cytokine response to PRRSV infection, and no significant difference was observed in cytokine expression compared to wild-type PAMs. Taken together, these data suggest that CD163 SRCR5-edited pigs are resistant to PRRSV 2, providing a basis for the establishment of PRRSV-resistant pig lines for commercial application and further investigation of the essential region of SRCR5 involved in virus infection.

5.
BMC Mol Cell Biol ; 20(1): 4, 2019 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-31041890

RESUMO

The white coat colour of Yorkshire and Landrace pig breeds is caused by the dominant white I allele of KIT, associated with 450-kb duplications and a splice mutation (G > A) at the first base in intron 17. To test whether genome editing can be employed to correct this structural mutation, and to investigate the role of KIT in the control of porcine coat colour, we designed sgRNAs targeting either intron 16 or intron 17 of KIT, and transfected Cas9/sgRNA co-expression plasmids into the kidney cells of Yorkshire pigs. The copy number of KIT was reduced by about 13%, suggesting the possibility of obtaining cells with corrected structural mutations of the KIT locus. Using single cell cloning, from 24 successfully expanded single cell clones derived from cells transfected with sgRNA targeting at intron 17, we obtained 3 clones with a single copy of KIT without the splice mutation. Taken together, the 12.5% (3/24) efficiency of correction of structural mutations of 450 kb fragments is highly efficient, providing a solid basis for the generation of genome edited Yorkshire pigs with a normal KIT locus. This provides an insight into the underlying genetic mechanisms of porcine coat colour.

6.
FASEB J ; 33(8): 9638-9655, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31145867

RESUMO

Here, we performed whole-genome bisulfite sequencing of longissimus dorsi muscle from Landrace and Wuzhishan (WZS) miniature pigs during 18, 21, and 28 d postcoitum. It was uncovered that in regulatory regions only around transcription start sites (TSSs), gene expression and methylation showed negative correlation, whereas in gene bodies, positive correlation occurred. Furthermore, earlier myogenic gene demethylation around TSSs and earlier hypermethylation of myogenic genes in gene bodies were considered to trigger their earlier expression in miniature pigs. Furthermore, by analyzing the methylation pattern of the myogenic differentiation 1(MyoD) promoter and distal enhancer, we found that earlier demethylation of the MyoD distal enhancer in WZSs contributes to its earlier expression. Moreover, DNA demethylase Tet1 was found to be involved in the demethylation of the myogenin promoter and promoted immortalized mouse myoblast cell line (C2C12) and porcine embryonic myogenic cell differentiation. This study reveals that earlier demethylation of myogenic genes contributes to precocious terminal differentiation of myoblasts in miniature pigs.-Zhang, X., Nie, Y., Cai, S., Ding, S., Fu, B., Wei, H., Chen, L., Liu, X., Liu, M., Yuan, R., Qiu, B., He, Z., Cong, P., Chen, Y., Mo, D. Earlier demethylation of myogenic genes contributes to embryonic precocious terminal differentiation of myoblasts in miniature pigs.

7.
Theriogenology ; 132: 95-105, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31004879

RESUMO

The EZH2 protein endows the polycomb repressive complex 2 (PRC2) with histone lysine methyltransferase activity that is associated with transcriptional repression. Recent investigations have documented crucial roles for EZH2 in mediating X-inactivation, stem cell pluripotency and cancer metastasis. However, there is little evidence demonstrating the maternal effect of EZH2 on porcine preimplantation development. Here, we took parthenogenetic activation embryos to eliminate the confounding paternal influence. We showed that the dynamic expression of EZH2 during early development was accompanied by changes in H3K27me3 levels. Depletion of EZH2 in MII oocytes by small interfering RNA not only impaired embryonic development at the blastocyst stage (P < 0.05), but also disrupted the equilibrium of H3K4me3 and H3K27me3 in the embryo. Interestingly, the expression of TET1, a member of Ten-Eleven Translocation gene family for converting 5-methylcytosine (5 mC) to 5-hydroxymethylcytosine (5hmC), was decreased after EZH2 knockdown, in contrast to the increase of the other two members, TET2 and TET3 (P < 0.05). These results indicate a correlation between histone methylation and DNA methylation, and between EZH2 and TET1. Along with the downregulation of TET1, the expression of the pluripotency gene NANOG was decreased (P < 0.05), which is consistent with a previous finding in mouse ES cells. Meanwhile, the abundance of OCT4 and SOX2 were also down-regulated. Moreover, EZH2 knockdown reduced the capacity of cells in the blastocysts to resist apoptosis. Taken together, our data suggest that EZH2 is integral to the developmental program of porcine parthenogenetic embryos and exerts its function by regulating pluripotency, differentiation and apoptosis.


Assuntos
Blastocisto/fisiologia , Desenvolvimento Embrionário/genética , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Técnicas de Silenciamento de Genes/veterinária , Partenogênese , Suínos/embriologia , Animais , Regulação da Expressão Gênica no Desenvolvimento , Suínos/genética
8.
J Agric Food Chem ; 67(16): 4700-4708, 2019 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-30929441

RESUMO

Fat-related traits have great influences on pork quality. As different fat tissues have different biochemical profiles depending on their location, intramuscular fat contributes to gustatory qualities, while subcutaneous fat is considered as a negative factor associated with growth performance. In this study, both primary intramuscular and subcutaneous vascular stem cells (IVSCs and SVSCs) could be differentiated into mature adipocytes, though the IVSC differentiation efficiency was lower. By comparative analysis of transcriptomes, 2524 differentially expressed genes (DEGs) were found between two VSCs before differentiation, while only 551 DEGs were found and enriched in two pathways including biosynthesis of unsaturated fatty acids after differentiation. This result indicated that differentiated VSCs were more similar. During differentiation, more DEGs existed in IVSCs than that in SVSCs, suggesting that adipogenesis of IVSCs might be more complex. Additionally, the expression level of DEGs involved in the adipogenic process helps to explain the difference of differentiation efficiency between IVSCs and SVSCs.


Assuntos
Adipócitos/citologia , Adipogenia , Células-Tronco/citologia , Gordura Subcutânea/citologia , Adipócitos/metabolismo , Animais , Ácidos Graxos Insaturados/biossíntese , Perfilação da Expressão Gênica , Carne Vermelha/análise , Células-Tronco/metabolismo , Gordura Subcutânea/irrigação sanguínea , Gordura Subcutânea/metabolismo , Suínos , Transcriptoma
9.
Transgenic Res ; 28(1): 141-150, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30488155

RESUMO

Insulin-like growth factor 2 (IGF2) plays an important role in the development of the foetus and in post-natal growth and development. A SNP within intron 3 of porcine IGF2 disrupts a binding site for the repressor, zinc finger BED-type containing 6 (ZBED6), leading to up-regulation of IGF2 in skeletal muscle and major effects on muscle growth, heart size, and fat deposition. This favourable mutation is common in Western commercial pig populations, but is not present in most indigenous Chinese pig breeds. Here, we described the efficient disruption of the ZBED6 binding site motif in intron 3 of IGF2 by CRISPR/Cas9 in porcine embryonic fibroblasts (PEFs) from the indigenous Chinese pig breed, Liang Guang Small Spotted pig. Disruption of the binding motif led to a drastic up-regulation of IGF2 expression in PEFs and enhanced myogenic potential and cell proliferation of PEFs. IGF2-edited pigs were then generated using somatic cell nuclear transfer. Enhanced muscle development was evident in one pig with biallelic deletion of the ZBED6 binding site motif, implying that the release of ZBED6 repression has a major effect on porcine muscle development. Our study confirmed the important effect of a mutation in the ZBED6 binding site motif on IGF2 expression and myogenesis, thus providing the basis for breeding a new line of Liang Guang Small Spotted pigs with improved lean meat percentage, a trait of great commercial value to pig producers.


Assuntos
Sistemas CRISPR-Cas/genética , Fator de Crescimento Insulin-Like II/genética , Desenvolvimento Muscular/genética , Proteínas Repressoras/genética , Dedos de Zinco , Alelos , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Cruzamento , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Íntrons/genética , Carne , Suínos
10.
Clin Genet ; 95(3): 409-414, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30474133

RESUMO

Premature ovarian insufficiency (POI) is a group of heterogeneous disorders characterized by decreased ovarian reserve and increased follicle stimulating hormone (FSH) levels. It is rarely associated with short stature. FIGLA mutations with POI are identified with regard to heterozygosity; till date, only one affected family has been identified with homozygous mutations in FIGLA but without functional evaluation. Here, we described two POI patients from a consanguineous family from China. An 18-year-old girl and her sister presented with primary amenorrhea and increased FSH and luteinizing hormone levels, but the sister also presented with short stature and bone age delay. Whole-genome sequencing analysis identified a recurrent homozygous mutation in the FIGLA gene, c.2 T > C (p.Met1Thr), in this family member with POI; this variant was segregated within the pedigree. This change was absent in 382 control subjects, and we did not detect any mutations in 39 other idiopathic POI patients. in vitro functional analysis indicates that the p.Met1Thr mutation does not affect the transcription of the FIGLA gene, but blocks the synthesis of the full-length FIGLA protein. Our results support the notion that bi-allelic recessive loss-of-function effects of FIGLA contribute to POI patients with short stature and expand the FIGLA-related phenotypic spectrum.

11.
BMC Biotechnol ; 18(1): 66, 2018 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-30340581

RESUMO

BACKGROUND: Targeted DNA integration is widely used in basic research and commercial applications because it eliminates positional effects on transgene expression. Targeted integration in mammalian cells is generally achieved through a double crossover event between the genome and a linear donor containing two homology arms flanking the gene of interest. However, this strategy is generally less efficient at introducing larger DNA fragments. Using the homology-independent NHEJ mechanism has recently been shown to improve efficiency of integrating larger DNA fragments at targeted sites, but integration through this mechanism is direction-independent. Therefore, developing new methods for direction-dependent integration with improved efficiency is desired. RESULTS: We generated site-specific double-strand breaks using ZFNs or CRISPR/Cas9 in the human CCR5 gene and a donor plasmid containing a 1.6-kb fragment homologous to the CCR5 gene in the genome. These DSBs efficiently drove the direction-dependent integration of 6.4-kb plasmids into the genomes of two human cell lines through single-crossover recombination. The integration was direction-dependent and resulted in the duplication of the homology region in the genome, allowing the integration of another copy of the donor plasmid. The CRISPR/Cas9 system tended to disrupt the sgRNA-binding site within the duplicated homology region, preventing the integration of another plasmid donor. In contrast, ZFNs were less likely to completely disrupt their binding sites, allowing the successive integration of additional plasmid donor copies. This could be useful in promoting multi-copy integration for high-level expression of recombinant proteins. Targeted integration through single crossover recombination was highly efficient (frequency: 33%) as revealed by Southern blot analysis of clonal cells. This is more efficient than a previously described NHEJ-based method (0.17-0.45%) that was used to knock in an approximately 5-kb long DNA fragment. CONCLUSION: We developed a method for the direction-dependent integration of large DNA fragments through single crossover recombination. We compared and contrasted our method to a previously reported technique for the direction-independent integration of DNA cassettes into the genomes of cultured cells via NHEJ. Our method, due to its directionality and ability to efficiently integrate large fragments, is an attractive strategy for both basic research and industrial application.


Assuntos
Sistemas CRISPR-Cas , Marcação de Genes/métodos , Troca Genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Genoma , Humanos , Plasmídeos/genética , Receptores CCR5/genética
12.
J Agric Food Chem ; 65(30): 6317-6328, 2017 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-28673084

RESUMO

The adipocyte differentiation process, controlled by a tightly regulated transcriptional cascade, contributes partly to determine intramuscular adipose tissue (IMAT) mass, which is associated with meat quality in food animals, as well as obesity and related metabolic complications in human. Thus, this study aimed to characterize genes critical for intramuscular preadipocyte differentiation. Primary intramuscular preadipocytes were isolated from pigs, and mRNA profiles were performed at several key points (0 h, 4 h, 8 h, 1 day, 2 days, and 6 days) during adipogenesis using microarrays. By gene functional analysis, we identified numerous differentially expressed genes among distinct stages of intramuscular preadipocyte differentiation, which included numbers of transcription factors in the early stages. We obtained 4 clusters of differential gene expression pattern, including crucial candidate genes associated with adipogenesis of intramuscular adipocytes. Further, we demonstrated that POSTN and FGFR4 suppressed, whereas AKR1CL1 promoted, the expression of adipogenic marker PPARγ and C/EBPα. Taken together, our data delineated the transcriptome landscape during porcine intramuscular preadipocyte differentiation, which provided a valuable resource for finding the genes responsible for IMAT formation.


Assuntos
Adipócitos/citologia , Adipogenia , Músculo Esquelético/metabolismo , Suínos/genética , Transcriptoma , Adipócitos/metabolismo , Animais , Proteínas/genética , Proteínas/metabolismo , Suínos/fisiologia
13.
Hum Reprod ; 32(4): 944-953, 2017 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-28175319

RESUMO

Empty follicle syndrome (EFS) is a reproductive disorder in which no oocytes are retrieved during IVF. The existence of genuine EFS (GEFS) is still controversial, and to date, only one missense mutation of Luteinizing Hormone/Choriogonadotropin Receptor (LHCGR) has been reported to be associated with this disease. Here, we describe a GEFS patient in a non-consanguineous family from China. A 27-year-old woman presented with a 5-year history of primary infertility and LH resistance-like ovaries of unequal sizes, but with normal levels of circulating LH. In spite of a satisfactory ovarian reserve and response, no oocytes were retrieved after two cycles of IVF. Her condition did not appear to be failure of the hCG injection. It is more likely to be a genetic cause. A novel homozygous mutation in LHCGR gene, c.1345G>A (p.Ala449Thr), was detected in this patient. Each of her parents is heterozygous for this change, and the change was absent from 407 control subjects. Alanine at this amino acid position was highly conserved and replacement of threonine was predicted to disrupt the third transmembrane helix of the rhodopsin-like G protein-coupled receptor domain. Protein localization studies revealed that a portion of the mutant LHCGR protein molecules was retained intracellularly. Signalling studies demonstrated that this mutation had differing effects on the response of LHCGR to hCG or LH at different concentrations. Specifically, at a concentration <1 IU/ml, the mutant was activated by hCG stimulation but partially resistant to LH stimulation; at a higher concentration (>1 IU/ml), the mutant was activated by both hCG and LH. These data suggest that screening for mutations in the LHCGR gene may assist in the diagnosis of patients with GEFS. The literature describing the relationship between phenotype and genotypes in females is reviewed, and possible aetiologies and treatment options for this disease are proposed based on our and other studies.


Assuntos
Infertilidade Feminina/genética , Doenças Ovarianas/genética , Receptores do LH/genética , Substituição de Aminoácidos , Feminino , Estudos de Associação Genética , Humanos , Infertilidade Feminina/patologia , Recuperação de Oócitos , Oócitos/crescimento & desenvolvimento , Estrutura Terciária de Proteína , Receptores do LH/química , Adulto Jovem
14.
Yi Chuan ; 39(1): 48-55, 2017 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-28115305

RESUMO

As Chinese have raised most pigs and consumed most pig products in the world, improving the fertility of sow is of economic benefits to the pig industry in China. The sheep BMP15 (bone morphogenetic protein 15) gene has been identified as a major gene for controlling ovulation rates and prolific traits, which are key factors affecting the fertility of livestock. As similar natural occurring mutations in the porcine BMP15 gene have not yet been reported, we speculated that introducing the same prolific sheep mutations into the porcine BMP15 gene by using the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system.


Assuntos
Proteína Morfogenética Óssea 15/genética , Sistemas CRISPR-Cas/genética , Proteínas RGS/genética , Animais , Marcação de Genes/métodos , Engenharia Genética/métodos , Mutação/genética , Suínos
15.
Biotechnol Lett ; 39(3): 351-358, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27858321

RESUMO

OBJECTIVES: To develop an in vitro method for rapid evaluation of the capability of a designed single guide RNAs (sgRNAs) to guide Cas9 nucleases to cleave target loci in mammalian cells. RESULTS: We constructed a Cas9/sgRNA plasmid with two SP6 promoters to simultaneously express Cas9 nuclease and the sgRNA and a negative selection plasmid harbouring a target site of the sgRNA. After co-transforming chemically competent E. coli DH5α cells with the two plasmids, the transformants were plated at a low density on two LB plates: one containing only ampicillin and the other containing both ampicillin and chloramphenicol. The colony-count on the ampicillin + chloramphenicol plate was compared with that on the ampicillin-only plate to calculate the survival percentage. The survival % was negatively correlated with the genome editing efficiency of the sgRNA in mammalian cells evaluated by a T7 endonuclease 1 (T7E1) assay (r ranged from -0.8 to -0.92). This system eliminates the need for cell culture, transfection, FACS sorting, PCR and T7E1 nuclease treatment, and significantly reduces the cost of screening for active sgRNAs, especially in the case of large-scale screening. CONCLUSIONS: We have developed a bacterial-based negative selection system for rapid screening of active sgRNAs in mammalian cells at a very low cost.


Assuntos
Escherichia coli/genética , Testes Genéticos/métodos , RNA Guia/genética , Animais , Sequência de Bases , Células HEK293 , Humanos , Mutação INDEL/genética , Camundongos , Plasmídeos/genética , RNA Guia/metabolismo , Sus scrofa
16.
Springerplus ; 5(1): 814, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27390654

RESUMO

BACKGROUND: Genome editors such as CRISPR/Cas9 and TALENs are at the forefront of research into methodologies for targeted modification of the mammalian genome. To date few comparative studies have been carried out to investigate the difference of genome editing characteristics between CRISPR/Cas9 and TALENs. While the CRISPR/Cas9 system has overtaken TALENs as the tool of choice for most research groups working in this field, we hypothesized that there could be certain applications whereby the application of TALENs would have specific benefits. Here we compare CRISPR/Cas9 and TALEN as tools for introducing site-specific editing events at an integrated EGFP gene in the genome of HEK293FT cells. RESULTS: Guide RNAs and TALEN pairs were designed to target two loci within the EGFP gene. We found that paired Cas9 nucleases induced targeted genomic deletion more efficiently and precisely than two TALEN pairs. However, when concurrently supplied with a plasmid template spanning the two DNA double-strand breaks (DSBs) within EGFP, TALENs stimulated homology directed repair (HDR) more efficiently than CRISPR/Cas9 and caused fewer targeted genomic deletions. CONCLUSIONS: Our data suggest that the choice of genome editing tool should be determined by the desired genome editing outcome. Such a rational approach is likely to benefit research outputs for groups working in fields as diverse as modification of cell lines, to animal models for disease studies, or gene therapy strategies.

17.
J Biotechnol ; 221: 49-54, 2016 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-26778541

RESUMO

Genome editors are powerful tools that allow modification of the nuclear DNA in eukaryotic cells both in vitro and in vivo. In vitro modified cells are often phenotypically indistinguishable from unmodified cells, hampering their isolation for analysis. Episomal reporters encoding fluorescent proteins can be used for enrichment of modified cells by flow cytometry. Here we compare two surrogate reporters, RGS and SSA, for the enrichment of porcine embryonic fibroblasts containing mutations induced by ZFNs or CRISPR/Cas9. Both systems were effective for enrichment of edited porcine cells with the RGS reporter proving more effective than the SSA reporter. We noted a higher-fold enrichment when editing events were induced by Cas9 compared to those induced by ZFNs, allowing selection at frequencies as high as 70%.


Assuntos
Fibroblastos/citologia , Genes Reporter , Mutação , Animais , Sistemas CRISPR-Cas , Embrião de Mamíferos/citologia , Genoma , Suínos
18.
Sheng Wu Gong Cheng Xue Bao ; 32(10): 1455-1464, 2016 Oct 25.
Artigo em Chinês | MEDLINE | ID: mdl-29027454

RESUMO

A mammalian nonclassical secretion sequence derived from mouse Engrailed2 homeoprotein (En2) was used to direct the secretion of the enhanced green fluorescent protein from Pichia pastoris. This signal peptide conferred the transport of enhanced green fluorescent protein into periplasm through an endoplasmic reticulum-golgi independent pathway, without inducing severe unfolded protein response as compared with Saccharomyces cerevisae α-factor preprosequence. This study implies that this mammalian nonclassical signal peptide could be developed as a useful tool for delivering cargoes to the cell surface of yeast.


Assuntos
Parede Celular , Proteínas de Fluorescência Verde/metabolismo , Sinais Direcionadores de Proteínas , Proteínas Recombinantes/metabolismo , Animais , Retículo Endoplasmático , Complexo de Golgi , Camundongos , Peptídeos , Pichia , Transporte Proteico , Saccharomyces cerevisiae , Resposta a Proteínas não Dobradas
19.
J Biotechnol ; 214: 69-74, 2015 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-26200831

RESUMO

Production of nuclear donor cells with a high percentage of desired modifications is a critical step in the successful generation of genetically modified pigs through somatic cell nuclear transfer (SCNT). The CRISPR/Cas9 system has been used for efficient modification of the nuclear DNA in eukaryotic cells, including porcine cells. However, in vitro modified cells are often phenotypically indistinguishable from unmodified cells, hampering their enrichment. Here we investigate a dual fluorescence selection system for the efficient enrichment of porcine embryonic fibroblasts (PEFs) with CRISPR/Cas9-induced chromosomal deletions. Enrichment of cells with 170 bp deletions reached a frequency of 74%, whilst enrichment of cells with a larger 5 kb deletions achieved a frequency of 46%. This demonstrates the utility of a dual fluorescence reporter as an attractive tool for improving the efficiency of generating genome edited pigs.


Assuntos
Sistemas CRISPR-Cas/genética , Citometria de Fluxo/métodos , Deleção de Genes , Engenharia Genética/métodos , Proteínas de Fluorescência Verde/genética , Animais , Sequência de Bases , Proteína Morfogenética Óssea 15/química , Proteína Morfogenética Óssea 15/genética , Proteína Morfogenética Óssea 15/metabolismo , Células Cultivadas , DNA/genética , Fibroblastos , Genoma/genética , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Dados de Sequência Molecular , Suínos
20.
Antivir Ther ; 20(6): 573-82, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25409681

RESUMO

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) causes severe diseases affecting the swine industry worldwide. Currently, no vaccination regimen has proven sustained success in preventing PRRSV infection. Therefore, there is an urgent need for new antiviral strategies to control PRRSV. In this study, the inhibitory effects and molecular mechanism of antimicrobial peptide protegrin-1 (PG-1) isolated from porcine leukocytes against PRRSV were evaluated in vitro. METHODS: Marc-145 cells or porcine alveolar macrophages (PAMs) were infected with PRRSV in the presence or absence of PG-1 for 36 h. The inhibitory effects of PG-1 were assessed by measuring the transcript and protein level of PRRSV ORF7 in cells and virus titres in the supernatants. Virus attachment and entry assays were performed to explore the molecular mechanism of PG-1 action. RESULTS: We demonstrated that PG-1 strongly inhibited PRRSV infection and replication by suppressing virus RNA and protein synthesis, virus progeny production and viral particles release. Furthermore, in the PRRSV life cycle, PG-1 mainly blocked viral attachment in Marc-145 cells. However, in PAMs, PG-1 could neither inhibit PRRSV replication nor elevate antiviral cytokine expression. CONCLUSIONS: Our findings for the first time show that PG-1 is an antiviral peptide with effective inhibitory effects on PRRSV infection and replication in Marc-145 cells.


Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Antivirais/farmacologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/efeitos dos fármacos , RNA Mensageiro/antagonistas & inibidores , Proteínas Virais/antagonistas & inibidores , Animais , Linhagem Celular Transformada , Relação Dose-Resposta a Droga , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Células Epiteliais/virologia , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Rim/efeitos dos fármacos , Rim/imunologia , Rim/virologia , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/virologia , Especificidade de Órgãos , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/metabolismo , Cultura Primária de Células , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Suínos , Proteínas Virais/genética , Proteínas Virais/metabolismo , Vírion/efeitos dos fármacos , Vírion/fisiologia , Ligação Viral/efeitos dos fármacos , Internalização do Vírus , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética
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