scholarly journals Efficient TALEN-mediated gene knockin at the bovine Y chromosome and generation of a sex-reversal bovine

2021 ◽  
Author(s):  
Ming Wang ◽  
ZhaoLin Sun ◽  
Fangrong Ding ◽  
Haiping Wang ◽  
Ling Li ◽  
...  
Keyword(s):  
Y Chromosome ◽  
Nuclear Transfer ◽  
Sex Reversal ◽  
Practical Application ◽  
Transcription Activator ◽  
New Methods ◽  
Fetal Fibroblasts ◽  
Male Phenotype ◽  
Solid Foundation ◽  
First Time

AbstractFunctional elucidation of bovine Y-chromosome genes requires available genome editing technologies. Meanwhile, it has yet to be proven whether the bovine Sry gene is the main or single factor involved in the development of the male phenotype in bovine. Here, we efficiently knocked out four Y-linked genes (Sry, ZFY, DDX3Y, and EIF2S3Y) in bovine fetal fibroblasts (BFFs) with transcription activator-like effector nucleases (TALENs) individually. Furthermore, we used TALEN-mediated gene knockin at the Sry gene and generated a sex-reversal bovine by somatic cell nuclear transfer (SCNT). The resulting bovine had only one ovary and was sterile. We demonstrate, for the first time, that the Sry gene is an important sex-determining gene in bovine. Our method lays a solid foundation for detecting the biology of the bovine Y chromosome, as it may provide an alternative biological model system for the study of mammalian sex determination, and new methods for the practical application in agricultural, especially for sex predetermination.

scholarly journals Uncovering dynamic evolution in the plastid genome of seven Ligusticum species provides insights into species discrimination and phylogenetic implications

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Can Yuan ◽  
Xiufen Sha ◽  
Miao Xiong ◽  
Wenjuan Zhong ◽  
Yu Wei ◽  
...  
Keyword(s):  
Plastid Genome ◽  
Species Discrimination ◽  
Chloroplast Genomes ◽  
Genomic Arrangement ◽  
Phylogenetic Implications ◽  
Solid Foundation ◽  
Conserved Gene ◽  
Fine Resolution ◽  
Genome Scale ◽  
First Time

AbstractLigusticum L., one of the largest members in Apiaceae, encompasses medicinally important plants, the taxonomic statuses of which have been proved to be difficult to resolve. In the current study, the complete chloroplast genomes of seven crucial plants of the best-known herbs in Ligusticum were presented. The seven genomes ranged from 148,275 to 148,564 bp in length with a highly conserved gene content, gene order and genomic arrangement. A shared dramatic decrease in genome size resulted from a lineage-specific inverted repeat (IR) contraction, which could potentially be a promising diagnostic character for taxonomic investigation of Ligusticum, was discovered, without affecting the synonymous rate. Although a higher variability was uncovered in hotspot divergence regions that were unevenly distributed across the chloroplast genome, a concatenated strategy for rapid species identification was proposed because separate fragments inadequately provided variation for fine resolution. Phylogenetic inference using plastid genome-scale data produced a concordant topology receiving a robust support value, which revealed that L. chuanxiong had a closer relationship with L. jeholense than L. sinense, and L. sinense cv. Fuxiong had a closer relationship to L. sinense than L. chuanxiong, for the first time. Our results not only furnish concrete evidence for clarifying Ligusticum taxonomy but also provide a solid foundation for further pharmaphylogenetic investigation.

scholarly journals Defective Calmodulin-Mediated Nuclear Transport of the Sex-Determining Region of the Y Chromosome (SRY) in XY Sex Reversal

2005 ◽  
Vol 19 (7) ◽  
pp. 1884-1892 ◽  
Author(s):  
Helena Sim ◽  
Kieran Rimmer ◽  
Sabine Kelly ◽  
Louisa M. Ludbrook ◽  
Andrew H. A. Clayton ◽  
...  
Keyword(s):  
Y Chromosome ◽  
Nuclear Import ◽  
High Mobility ◽  
Sex Reversal ◽  
High Mobility Group ◽  
Missense Mutations ◽  
Nuclear Localization Signals ◽  
Xy Sex Reversal

Abstract The sex-determining region of the Y chromosome (SRY) plays a key role in human sex determination, as mutations in SRY can cause XY sex reversal. Although some SRY missense mutations affect DNA binding and bending activities, it is unclear how others contribute to disease. The high mobility group domain of SRY has two nuclear localization signals (NLS). Sex-reversing mutations in the NLSs affect nuclear import in some patients, associated with defective importin-β binding to the C-terminal NLS (c-NLS), whereas in others, importin-β recognition is normal, suggesting the existence of an importin-β-independent nuclear import pathway. The SRY N-terminal NLS (n-NLS) binds calmodulin (CaM) in vitro, and here we show that this protein interaction is reduced in vivo by calmidazolium, a CaM antagonist. In calmidazolium-treated cells, the dramatic reduction in nuclear entry of SRY and an SRY-c-NLS mutant was not observed for two SRY-n-NLS mutants. Fluorescence spectroscopy studies reveal an unusual conformation of SRY.CaM complexes formed by the two n-NLS mutants. Thus, CaM may be involved directly in SRY nuclear import during gonadal development, and disruption of SRY.CaM recognition could underlie XY sex reversal. Given that the CaM-binding region of SRY is well-conserved among high mobility group box proteins, CaM-dependent nuclear import may underlie additional disease states.

European Mail Armour

2021 ◽  
Author(s):  
Martijn A. Wijnhoven
Keyword(s):  
Early Modern ◽  
Middle Ages ◽  
Iron Age ◽  
Early Modern Period ◽  
Vital Role ◽  
Military Technology ◽  
Roman Army ◽  
Solid Foundation ◽  
First Time

Mail armour (commonly mislabelled 'chainmail') was used for more than two millennia on the battlefield. After its invention in the Iron Age, mail rapidly spread all over Europe and beyond. The Roman army, keen on new military technology, soon adopted mail armour and used it successfully for centuries. Its history did not stop there and mail played a vital role in warfare during the Middle Ages up to the Early Modern Period. Given its long history, one would think mail is a well-documented material, but that is not the case. For the first time, this books lays a solid foundation for the understanding of mail armour and its context through time. It applies a long-term multi-dimensional approach to extract a wealth of as yet untapped information from archaeological, iconographic and written sources. This is complemented with technical insights on the mail maker’s chaîne opératoire.

Roles of Anti-Müllerian hormone (Amh) and its duplicates in sex determination and germ cell proliferation of Nile tilapia

Genetics ◽  
2021 ◽  
Author(s):  
Xingyong Liu ◽  
Shengfei Dai ◽  
Jiahong Wu ◽  
Xueyan Wei ◽  
Xin Zhou ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Sex Determination ◽  
Germ Cell ◽  
Y Chromosome ◽  
X Chromosome ◽  
Nile Tilapia ◽  
Critical Period ◽  
Sex Reversal ◽  
Homozygous Mutation ◽  
Germ Cell Proliferation

Abstract Duplicates of amh are crucial for fish sex determination and differentiation. In Nile tilapia, unlike in other teleosts, amh is located on X chromosome. The Y chromosome amh (amh△-y) is mutated with 5 bp insertion and 233 bp deletion in the coding sequence, and tandem duplicate of amh on Y chromosome (amhy) has been identified as the sex determiner. However, the expression of amh, amh△-y and amhy, their roles in germ cell proliferation and the molecular mechanism of how amhy determines sex is still unclear. In this study, expression and functions of each duplicate were analyzed. Sex reversal occurred only when amhy was mutated as revealed by single, double and triple mutation of the three duplicates in XY fish. Homozygous mutation of amhy in YY fish also resulted in sex reversal. Earlier and higher expression of amhy/Amhy was observed in XY gonads compared with amh/Amh during sex determination. Amhy could inhibit the transcription of cyp19a1a through Amhr2/Smads signaling. Loss of cyp19a1a rescued the sex reversal phenotype in XY fish with amhy mutation. Interestingly, mutation of both amh and amhy in XY fish or homozygous mutation of amhy in YY fish resulted in infertile females with significantly increased germ cell proliferation. Taken together, these results indicated that up-regulation of amhy during the critical period of sex determination makes it the sex-determining gene, and it functions through repressing cyp19a1a expression via Amhr2/Smads signaling pathway. Amh retained its function in controlling germ cell proliferation as reported in other teleosts, while amh△-y was nonfunctionalized.

Fertile females of the mole Talpa occidentalis are phenotypic intersexes with ovotestes

Development ◽  
1993 ◽  
Vol 118 (4) ◽  
pp. 1303-1311 ◽  
Author(s):  
R. Jimenez ◽  
M. Burgos ◽  
A. Sanchez ◽  
A.H. Sinclair ◽  
F.J. Alarcon ◽  
...  
Keyword(s):  
Y Chromosome ◽  
Sex Reversal ◽  
Chromosome Gene ◽  
Different Populations ◽  
Testis Determining Gene

We investigated the origin of XX sex reversal in the insectivorous mole Talpa occidentalis. Cytogenetic, histological and hormonal studies indicate that all XX individuals analyzed from two different populations are true hermaphrodites, with ovotestes. This suggests that XX sex reversal may be the norm in this species. The intersexes are functional fertile females and the trait is transmitted and maintained in the population. Intersexes lack the Y chromosome gene SRY (sex determining region Y gene), shown to be the testis determining gene. These results suggest that XX intersex moles may have arisen from a mutation of a gene located downstream from SRY/TDY in the testis determining pathway.

ONLINE EXHIBITION METROLEXPO-2020. ACCURATE MEASUREMENTS ARE THE BASIS OF QUALITY AND SAFETY

2021 ◽  
pp. 50-57
Author(s):  
V. I. Matveev
Keyword(s):  
Measuring Equipment ◽  
Technical Diagnostics ◽  
Quality And Safety ◽  
New Methods ◽  
Analytical Studies ◽  
Functional Control ◽  
First Time

The article summarizes the results of the MetrolExpo exhibition, which was held for the first time online. The event focused on instrumentation-demonstration and discussion of the possibilities of modern measuring equipment, analysis of new methods and technologies of accurate measurements that have appeared in recent years. The latest developments, devices and systems for conducting measurements, tests, technical diagnostics, analytical studies, production and functional control from the largest Russian and foreign manufacturers were demonstrated.

scholarly journals Dynamic Methylation Changes of DNA and H3K4 by RG108 Improve Epigenetic Reprogramming of Somatic Cell Nuclear Transfer Embryos in Pigs

2018 ◽  
Vol 50 (4) ◽  
pp. 1376-1397 ◽  
Author(s):  
Yanhui Zhai ◽  
Zhiren Zhang ◽  
Hao Yu ◽  
Li Su ◽  
Gang Yao ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Histone Modifications ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Histone H3 ◽  
Dna Demethylation ◽  
Epigenetic Reprogramming ◽  
Expression Levels ◽  
Fetal Fibroblasts

Background/Aims: DNA methylation and histone modifications are essential epigenetic marks that can significantly affect the mammalian somatic cell nuclear transfer (SCNT) embryo development. However, the mechanisms by which the DNA methylation affects the epigenetic reprogramming have not been fully elucidated. Methods: In our study, we used quantitative polymerase chain reaction (qPCR), Western blotting, immunofluorescence staining (IF) and sodium bisulfite genomic sequencing to examine the effects of RG108, a DNA methyltransferase inhibitor (DNMTi), on the dynamic pattern of DNA methylation and histone modifications in porcine SCNT embryos and investigate the mechanism by which the epigenome status of donor cells’ affects SCNT embryos development and the crosstalk between epigenetic signals. Results: Our results showed that active DNA demethylation was enhanced by the significantly improving expression levels of TET1, TET2, TET3 and 5hmC, and passive DNA demethylation was promoted by the remarkably inhibitory expression levels of DNMT1, DNMT3A and 5mC in embryos constructed from the fetal fibroblasts (FFs) treated with RG108 (RG-SCNT embryos) compared to the levels in embryos from control FFs (FF-SCNT embryos). The signal intensity of histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 9 acetylation (H3K9Ac) was significantly increased and the expression levels of H3K4 methyltransferases were more than 2-fold higher expression in RG-SCNT embryos. RG-SCNT embryos had significantly higher cleavage and blastocyst rates (69.3±1.4%, and 24.72±2.3%, respectively) than FF-SCNT embryos (60.1±2.4% and 18.38±1.9%, respectively). Conclusion: Dynamic changes in DNA methylation caused by RG108 result in dynamic alterations in the patterns of H3K4me3, H3K9Ac and histone H3 lysine 9 trimethylation (H3K9me3), which leads to the activation of embryonic genome and epigenetic modification enzymes associated with H3K4 methylation, and contributes to reconstructing normal epigenetic modifications and improving the developmental efficiency of porcine SCNT embryos.

scholarly journals TYPICAL CLASS METHODS FOR SOLVING EQUATIONS AND INEQUALITIES WITH DIFFERENT STRUCTURES

2021 ◽  
Vol 58 (3) ◽  
pp. 53-62
Author(s):  
A.K. Alpysov ◽  
◽  
A.K. Seytkhanova ◽  
I.Sh. Abishova ◽  
◽  
...  
Keyword(s):  
Problem Solving ◽  
Mathematical Thinking ◽  
Mathematical Concept ◽  
Thinking Skills ◽  
Practical Application ◽  
Mathematical Concepts ◽  
Theoretical Substantiation ◽  
New Methods ◽  
General Article ◽  
Solving Equations

The article discusses the ways of developing skills and abilities to effectively solve problems when describing methods for solving equations and inequalities, clarifying theoretical knowledge, the basics of forming skills for practical application. The formation of mathematical concepts through solving problems in teaching mathematics opens the way to the development of mathematical thinking, the application of knowledge in practice, and the development of search skills. To master a mathematical concept, along with its definition, it is necessary to know its features and properties. This can be achieved primarily through problem solving and exercise. Problem solving is based on the development of new methods, the creation of algorithms, ways of developing practical skills in the methods and techniques mastered with the help of tasks.In addition, transforming equations and inequalities through the development of thinking skills helps to identify common or special properties in order to draw correct conclusions. Solving various problems, it shows what operations should be used to determine the situation in which a solution was found, and what features of the solution allow choosing the most effective methods. Thanks to the theoretical substantiation of the general article, it is possible to master convenient methods for solving equations and inequalities of various structures.

scholarly journals 63 IMPROVED IN VITRO DEVELOPMENT OF PORCINE EMBRYOS PRODUCED BY NUCLEAR TRANSFER, IVF AND PARTHENOGENESIS

2005 ◽  
Vol 17 (2) ◽  
pp. 181 ◽  
Author(s):  
D. Sage ◽  
P. Hassel ◽  
B. Petersen ◽  
W. Mysegades ◽  
P. Westermann ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Culture Media ◽  
Cumulus Cells ◽  
Cell Number ◽  
Foster Mother ◽  
Chi Square ◽  
Fetal Fibroblasts ◽  
Blastocyst Rate ◽  
Parthenogenetic Embryos

Porcine nuclear transfer (NT) is an inefficient process and it is necessary to use as many as 120 NT embryos for each foster mother to obtain small litters of live piglets. In these experiments, we evaluated the effects of culture atmosphere and medium on the development of NT embryos by monitoring blastocyst rate and cell number of Day 6 blastocysts. Age matched IVF and parthenogenetic embryos were also evaluated for comparison. For all experiments a pool of oocytes was aspirated from ovaries collected in a local abattoir. Following aspiration, oocytes were allowed to mature for 40 h in North Carolina State University (NCSU)-37 medium (supplemented with cAMP and hCG/eCG for the first 22 h). After removal of the cumulus cells, denuded oocytes with polar bodies were selected for NT, enucleated, fused with fetal fibroblasts, and sequentially activated electrically and chemically by 3 h of treatment with 6-dimethylaminopurine (6-DMAP). A second group of oocytes from the same denuded pool were maintained in TL-HEPES medium and activated in parallel with the NT group to produce parthenogenetic embryos. A third group was fertilized with frozen-thawed epididymal semen and co-cultured for ∼12 h to give IVF embryos. All three treatment groups were subdivided into a control subgroup and an experimental subgroup. In the first experiment, we compared the effects of atmosphere (20% vs. 5% oxygen) on in vitro embryonic development in NCSU-23 medium. In the second experiment, we used only the 5% oxygen concentration and compared different culture media. One subgroup was maintained in standard NCSU-23 medium and the second subgroup was cultured in a two-step system for the first 58 h in modified NCSU-23 (without glucose but supplemented with 2.0 mM lactate and 0.2 mM pyruvate), followed by addition of glucose to give a final concentration of 5.55 mM. Data were statistically analyzed by analysis of variance and chi square test. Blastocyst rate and mean cell number in all three embryo groups were improved under 5% oxygen. The most dramatic effect was observed in the NT group, in which the blastocyst rate increased significantly (P < 0.001) from 6.7% ± 5.9 (n = 279) to 19.6% ± 8.9 (n = 250) and mean cell number increased from 17.7 ± 12.1 to 25.8 ± 10.3 cells per blastocyst. With 5% oxygen there was also an increase of blastocyst rates and mean cell numbers in both IVF and parthenogenetic groups. In the second experiment, blastocyst rate for NT embryos increased significantly (P < 0.05) from 21.8% ± 7.6 (n = 242) in conventional NCSU-23 to 31.5% ± 11.0 (n = 271) in the modified system whereas there was almost no difference in the mean cell number of both groups (29.2 ± 4.3 vs. 31.5 ± 5.3). In the groups of IVF and parthenogenetic embryos no difference was found. These results indicate that both the reduced oxygen and the modified culture medium are important for pre-implantation development of porcine nuclear transfer embryos.

Generation of ETV5 Knockout Pigs with CRISPR/Cas9

2021 ◽  
Author(s):  
Mao Zhang ◽  
Gengyuan Cai ◽  
Rong Zhou ◽  
Huaqiang Yang
Keyword(s):  
Nuclear Transfer ◽  
Gene Editing ◽  
Seminiferous Tubules ◽  
Rt Pcr ◽  
Self Renewal ◽  
Progressive Loss ◽  
Elisa Testing ◽  
Fetal Fibroblasts ◽  
Normal Spermatogenesis ◽  
Hematoxylin And Eosin

Background: Ets variant factor 5 (ETV5) plays an important regulatory role in mouse Spermatogonial stem cells (SSCs) self-renewal. ETV5 knockout (KO) mice exhibit a progressive loss of SSCs and resulting in a Sertoli cell-only phenotype. The current study was aimed to use gene editing technology to obtain ETV5-KO pigs as a model for studying the apoptosis mechanism of SSCs and further clarify the function of ETV5 gene in pigs.Methods: A gene editing plasmid for the porcine ETV5 gene was constructed, transfected into porcine fetal fibroblasts by electroporation to obtain ETV5-KO cells. ETV5-KO cells were used as donors to prepare ETV5-KO pigs by somatic cell nuclear transfer (SCNT). Testis tissues were collected for hematoxylin and eosin (HE), immunohistochemistry (IHC), RT-PCR testing and blood for ELISA testing from ETV5-KO pig.Result: In the present study, we used the CRISPR/Cas9 system and SCNT to generate homozygous ETV5-KO pigs. We observed 3 phenotypes in these pigs: normal testis development after birth, the SSCs in the seminiferous tubules did not show obviously extinction at sexual maturity and normal spermatogenesis.

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