scholarly journals Correction of a CFTR/G542X Mutation Using CRISPR/Cas9 Editing in Ovine-bovine Interspecies Embryos

2021 ◽  
Author(s):  
Zhiqiang Fan ◽  
Ying Liu ◽  
Iuri Perisse ◽  
Ann Harris ◽  
Kenneth White ◽  
...  
Keyword(s):  
Transmembrane Conductance Regulator ◽  
Gene Correction ◽  
Human Genetic Disease ◽  
Cell Stage ◽  
Homology Directed Repair ◽  
Stage Embryo ◽  
Developmental Capacity ◽  
Embryo Stage ◽  
Cf Transmembrane Conductance Regulator

Abstract Cystic fibrosis (CF) is a human genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Correction of CFTR mutations at embryo stage could be a permanent solution to cure this disease. To assess the efficiency of CFTR/G542X mutation correction in vitro by CRISPR/Cas9, we utilized embryos generated by ovine-bovine interspecies somatic cell nuclear transfer (iSCNT) due to a limited access to sheep oocytes. First, we evaluated the developmental capacity of reconstructed iSCNT embryos. These embryos were able to develop to 16-cell stage, allowing for individual embryo genotyping. Then, we optimized the concentrations of Cas9:gRNA ribonucleoprotein (RNP) for 1-cell stage embryo injection. Genotyping results showed that we achieved high efficiencies (88.9–100%) of indel mutations at the target locus after injection of different concentrations of RNP. When an RNP (0.09 µg/µl:2.3 µM) was co-injected with a ssODN (18 µM), the G542X mutation was corrected via the homology-directed repair in 11.1% (1/9) of iSCNT embryos. Taken together, we developed an effective strategy to correct the CFTR/G542X mutation in ovine-bovine iSCNT embryos by CRISRP/Cas9. Our strategy overcomes the limitation of oocyte source and provides the opportunity of mimicking the editing of any other genes in one-cell embryos of different species.

92 Correction of the CFTR G542X mutation using CRISPR/Cas9 genome editing in ovine-bovine interspecies embryos

2021 ◽  
Vol 33 (2) ◽  
pp. 153
Author(s):  
Z. Fan ◽  
Y. Liu ◽  
I. V. Perisse ◽  
K. L. White ◽  
I. A. Polejaeva
Keyword(s):  
Cftr Gene ◽  
Transmembrane Conductance Regulator ◽  
Human Genetic Disease ◽  
Cell Stage ◽  
Homology Directed Repair ◽  
Guide Rnas ◽  
Significant Difference ◽  
Cftr Mutations ◽  
Developmental Capacity ◽  
Cf Transmembrane Conductance Regulator

Cystic fibrosis (CF) is a human genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. We have recently generated 3 CF sheep models: a CFTR−/− model (Fan et al. 2018 LCI Insight 3:e123529; https://doi.org/10.1172/jci.insight.123529) and 2 additional models where we introduced human G542X and F508del mutations into the sheep genome (unpublished). Correction of CFTR mutations in zygotes with gene-editing techniques could be a permanent solution to cure this disease. To assess the efficiency of mutation correction invitro by CRISPR/Cas9, we utilised embryos generated by ovine-bovine interspecies SCNT (iSCNT) due to limited access to sheep oocytes. First, we evaluated the developmental capacity of reconstructed iSCNT embryos, in which nucleus donors were derived from ovine fibroblasts and recipient cytoplasm from enucleated bovine oocytes. These iSCNT embryos were able to develop to 16- to 32-cell stage (3/30, 10.0%), which allowed the genotyping of each embryo using PCR-restriction fragment length polymorphism assays and Sanger sequencing. Then, specific single-guide RNAs (sgRNAs) and 101-bp single-stranded oligodeoxynucleotides (ssODNs) were designed and synthesised to correct the G542X mutation in the sheep CFTR gene. We optimized the concentrations of Cas9:sgRNA ribonucleoproteins (RNPs) for 1-cell stage embryonic injection. Mutation analysis of embryos was conducted at 3 days post injection. Genotyping results showed that we achieved high efficiencies (95.7–100%) of mutations (indels) at targeting loci after injection of different concentrations of Cas9:sgRNA RNPs (0.02 µg:0.6 pmol/µL to 1.4 µg:40 pmol/µL). Furthermore, when an RNP (1.4 µg:40 pmol/µL) was co-injected with a ssODN (80 pmol/µL), both targeting the G542X mutation, the mutation was successfully corrected in the genome of iSCNT embryos generated using G542X fibroblasts as nucleus donors at an efficiency of 5.7% (3/53) via homology-directed repair mechanism. During the invitro culture of iSCNT embryos, we did not observe significant difference (P>0.05, unpaired t-test) in cleavage rates between embryos with or without injection (85.5% vs. 89.0%). Off-target analysis of those mutated and G542X-corrected embryos is in progress. Our strategy overcomes the limitation of oocyte source and provides an opportunity to mimic the editing of any other gene in embryos of different species.

scholarly journals Baicalin improves the in vitro developmental capacity of pig embryos by inhibiting apoptosis, regulating mitochondrial activity and activating sonic hedgehog signaling

2019 ◽  
Vol 25 (9) ◽  
pp. 538-549 ◽  
Author(s):  
Qing Guo ◽  
Mei-Fu Xuan ◽  
Zhao-Bo Luo ◽  
Jun-Xia Wang ◽  
Sheng-Zhong Han ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Signaling Pathway ◽  
Sonic Hedgehog ◽  
Hedgehog Signaling ◽  
Control Group ◽  
Mitochondrial Activity ◽  
Cell Stage ◽  
Shh Signaling ◽  
Developmental Capacity

Abstract Baicalin, a traditional Chinese medicinal monomer whose chemical structure is known, can be used to treat female infertility. However, the effect of baicalin on embryonic development is unknown. This study investigated the effects of baicalin on in vitro development of parthenogenetically activated (PA) and in vitro fertilized (IVF) pig embryos and the underlying mechanisms involved. Treatment with 0.1 μg/ml baicalin significantly improved (P < 0.05) the in vitro developmental capacity of PA pig embryos by reducing the reactive oxygen species (ROS) levels and apoptosis and increasing the mitochondrial membrane potential (ΔΨm) and ATP level. mRNA and protein expression of sonic hedgehog (SHH) and GLI1, which are related to the SHH signaling pathway, in PA pig embryos at the 2-cell stage, were significantly higher in the baicalin-treated group than in the control group. To confirm that the SHH signaling pathway is involved in the mechanism by which baicalin improves embryonic development, we treated embryos with baicalin in the absence or presence of cyclopamine (Cy), an inhibitor of this pathway. Cy abolished the effects of baicalin on in vitro embryonic development. In conclusion, baicalin improves the in vitro developmental capacity of PA and IVF pig embryos by inhibiting ROS production and apoptosis, regulating mitochondrial activity and activating SHH signaling.

185 DEVELOPMENT CAPACITY OF PRE- AND POSTPUBERTAL PIG OOCYTES EVALUATED BY SOMATIC CELL NUCLEAR TRANSFER AND PARTHENOGENETIC ACTIVATION

2013 ◽  
Vol 25 (1) ◽  
pp. 241 ◽  
Author(s):  
H. S. Pedersen ◽  
R. Li ◽  
Y. Liu ◽  
P. Løvendahl ◽  
P. Holm ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cell Number ◽  
Time Interval ◽  
Cell Stage ◽  
Parthenogenetic Activation ◽  
Development Capacity ◽  
Developmental Capacity

Most of the porcine oocytes used for in vitro studies are collected from gilts. Our aims were to study development capacity of gilt v. sow oocytes (pre- and postpubertal respectively) using 2 techniques illustrating development competence [parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT)], and to describe a simple method to select the most competent oocytes. Inside-ZP diameter of in vitro-matured gilt oocytes was measured (µm; small ≤110; medium >110; large ≥120). Gilt and sow oocytes were morphologically grouped as good (even cytoplasm, smooth cell membrane, visible perivitelline space) or bad before used for PA (good and bad) or SCNT (good). The PA and SCNT were performed as before with minor modifications (Cryobiol. 64, 60; Cell. Reprogr. 13, 521) before culture for 6 days in a standard or timelapse incubator. Rates of cleavage (CL%, Day 2), blastocyst (BL%, Day 6), and blastocyst cell number (Hoechst 33342) were recorded. For PA embryos in a timelapse incubator (26 oocytes/group; 2 replicates), the first appearance of 2-cell stage was recorded. Between groups, CL% and BL% were analysed by chi-square and cell number by t-test. Results are presented in the table for the development of good oocytes after PA. The results show a low CL% of small-gilts compared with the other groups. The BL% increased with gilt-oocyte-diameter; however, sow oocytes reached the highest BL%. Total cell number was higher in sow than in gilt blastocysts. The SCNT experiments showed no differences in CL% (90–96) and blastocyst cell number (51–59) between groups. The BL% was higher in medium gilts and sows (41; 45) compared with large gilts (21). The BL% of bad oocytes was 1% from all 4 groups (176 oocytes, 25 replicates). Time interval for appearance of 2-cell stage for embryos developing into blastocysts showed no differences between groups (19–20 h). Within groups, this time interval showed a larger standard deviation for embryos not developing v. embryos developing into blastocysts. It is concluded that (a) sow oocytes have higher developmental capacity compared to gilts, (b) small gilt oocytes are not developmentally competent, (c) measurement of inside-ZP diameter, combined with morphological selection, is useful to remove non-competent oocytes. Further studies are needed to dissect the developmental capacity of medium and large gilt oocytes. Also, further timelapse studies may reveal a time interval in which the first cleavage of embryos with high developmental capacity takes place. Table 1.Rates of cleavage (CL%), blastocyst (BL%), and total no. of cells (mean ± SEM) in blastocysts of PA embryos from gilts and sows1

95 Invitro correction of F508del and G542X mutations in sheep fibroblasts of cystic fibrosis models

2021 ◽  
Vol 33 (2) ◽  
pp. 155
Author(s):  
K. Bunch ◽  
I. V. Perisse ◽  
Z. Fan ◽  
K. White ◽  
I. Polejaeva
Keyword(s):  
Cystic Fibrosis ◽  
Genetic Disease ◽  
The United States ◽  
Human Genetic Disease ◽  
Nonsense Mediated Decay ◽  
Homology Directed Repair ◽  
Mutant Transcript ◽  
Pcr Products ◽  
Exon 11 ◽  
Cf Transmembrane Conductance Regulator

Cystic fibrosis (CF) is a human genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Among the ∼2000 known CF mutations, the F508del mutation is found in 84% and G542X in 4.6% of the CF patients in the United States. The F508del mutation occurs in exon 11 and is characterised by deletion of the “CTT” nucleotides, resulting in deletion on the phenylalanine residue at the position 508 of CFTR. This causes misfolding of the CFTR protein, which is further degraded by proteases. The G542X mutation is a nonsense mutation found in exon 12 and associated with nonsense-mediated decay of the mutant transcript causing the absence of protein production. Previously, we generated CFTRF508del/F508del and CFTRG542X/G542X lambs (unpublished) using CRISPR/Cas9 and somatic cell nuclear transfer (SCNT) techniques. We hypothesised that gene editing may be an effective tool to correct these mutations and permanently cure this genetic disease. Thus, in this study, we evaluated the efficiency of CRISPR/Cas9-meditated gene knock-in to correct the F508del and G542X mutations in sheep fibroblasts invitro. We designed single guide (sg)RNAs using the Benchling software (https://benchling.com/academic) and approximately 100bp of single-stranded oligodeoxynucleotides (ssODNs) targeting the mutation sites at exon 11 and 12 to introduce either “CTT” or change the “T” to “G” nucleotide in genome of F508del or G542X CF sheep cells, respectively. Each of Cas9/sgRNA ribonucleoproteins was transfected into sheep fibroblast cells along with ssODNs using the Lonza-4D-NucleofectorTM (Lonza) system for homology-directed repair. The transfected cells were subsequently cultured in Dulbecco’s modified Eagle medium, supplemented with 15% fetal bovine serum and 1% penicillin, and incubated at 38.5°C. DNA was extracted 48h post-transfection to validate mutation efficiency. PCR products of the exons 11 and 12 were ligated into T-vector, and bacterial colonies were selected based on blue/white screening. In total, we isolated 32 single cell bacterial colonies for each mutant. Sequencing results indicate that “CTT” was introduced in 4/26 (15.3%) plasmid colonies, and “T to G” replaced in 13/31 (41.9%) colonies. Therefore, our results indicate that the F508del and G542X mutations can be effectively corrected in CF sheep fibroblasts invitro using a CRISPR/Cas9 approach.

Effect of maturation medium on in vitro cleavage of canine oocytes fertilized with fresh and cooled homologous semen

Zygote ◽  
2007 ◽  
Vol 15 (1) ◽  
pp. 43-53 ◽  
Author(s):  
B. de Ávila Rodrigues ◽  
L. Carboneiro dos Santos ◽  
J.L. Rodrigues
Keyword(s):  
Cell Expansion ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Final Concentration ◽  
Comparative Experiment ◽  
Cell Stage ◽  
Maturation Medium ◽  
Developmental Capacity ◽  
Fresh Semen

SUMMARYThis study evaluated the effect of three maturation media on the development of in vitro-matured and in vitro-fertilized dog oocytes. In Experiment 1 (non-comparative experiment) canine cumulus–oocyte complexes (COCs) were matured in vitro in TCM199 supplemented with estrous cow serum (10%) + gonadotropins + steroid (treatment A), TCM199 + estrous cow serum (10%) (treatment B), or TCM199 + polyvinylpyrrolidone (PVP) (4%) (treatment C). All maturation media contained a final concentration of 1 μg/ml of human somatotropin (hST). Oocytes were fertilized with fresh ejaculated sperm and development was assessed by cleavage. The objective of Experiment 2 (comparative experiment) was to compare the rates of cleavage and developmental capacity of COCs matured in vitro in same medium as in Experiment 1, and fertilized either with fresh ejaculated or with cooled extended homologous spermatozoa. In Experiments 1 and 2, oocytes fertilized with fresh semen were in vitro-matured for 48 h, while in Experiment 2 COCs fertilized with cooled semen were matured in vitro for 72 h. The results of Experiments 1 and 2 demonstrated that cleavage was not influenced by the oocyte's maturation environment. The results of Experiment 1 showed that pronucleus formation + cleavage (day 7 after IVF) was similar among treatments A, B and C (p = 0.277). Also, in Experiment 2, pronucleus formation + cleavage (day 7 after IVF) was not different for oocytes fertilized in vitro either with fresh or cooled semen and maturated in media A (p = 0.190), B (p = 0.393) or C (p = 0.687). In both experiments, the numbers of embryos that developed to the 6–8-cell stage were higher for oocytes matured in medium A and fertilized with fresh semen, when compared with numbers of oocytes matured in media B and C. Embryo development to the 6–8-cell stage of oocytes fertilized either with fresh or cooled sperm was observed in treatments A and C in Experiment 2. Cumulus cell expansion was similar among treatments in Experiment 1. In Experiment 2, cumulus cell expansion among treatments A, B and C was similar after 48 h or 72 h of IVM. In both experiments, the greatest expansion category seen was for category 2 (outer cumulus cells slightly expanded). No correlation between cumulus expansion and cleavage were observed. Polyspermy rates in oocytes matured in medium A, and fertilized with fresh sperm were not significantly different from polyspermy rates observed using media B and C, in both experiments. Our findings indicate that treatments A, B and C are similarly effective for the cleavage of dog oocytes. Furthermore, it was demonstrated that canine oocytes matured in vitro could be fertilized by homologous cooled spermatozoa and progress to cleavage.

scholarly journals Cooh-Terminal Truncations Promote Proteasome-Dependent Degradation of Mature Cystic Fibrosis Transmembrane Conductance Regulator from Post-Golgi Compartments

2001 ◽  
Vol 153 (5) ◽  
pp. 957-970 ◽  
Author(s):  
Mohamed Benharouga ◽  
Martin Haardt ◽  
Norbert Kartner ◽  
Gergely L. Lukacs
Keyword(s):  
Cystic Fibrosis ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Cell Surface Biotinylation ◽  
Partially Unfolded ◽  
Cf Transmembrane Conductance Regulator

Impaired biosynthetic processing of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel, constitutes the most common cause of CF. Recently, we have identified a distinct category of mutation, caused by premature stop codons and frameshift mutations, which manifests in diminished expression of COOH-terminally truncated CFTR at the cell surface. Although the biosynthetic processing and plasma membrane targeting of truncated CFTRs are preserved, the turnover of the complex-glycosylated mutant is sixfold faster than its wild-type (wt) counterpart. Destabilization of the truncated CFTR coincides with its enhanced susceptibility to proteasome-dependent degradation from post-Golgi compartments globally, and the plasma membrane specifically, determined by pulse–chase analysis in conjunction with cell surface biotinylation. Proteolytic cleavage of the full-length complex-glycosylated wt and degradation intermediates derived from both T70 and wt CFTR requires endolysosomal proteases. The enhanced protease sensitivity in vitro and the decreased thermostability of the complex-glycosylated T70 CFTR in vivo suggest that structural destabilization may account for the increased proteasome susceptibility and the short residence time at the cell surface. These in turn are responsible, at least in part, for the phenotypic manifestation of CF. We propose that the proteasome-ubiquitin pathway may be involved in the peripheral quality control of other, partially unfolded membrane proteins as well.

scholarly journals Transposable elements drive reorganisation of 3D chromatin during early embryogenesis

2019 ◽  
Author(s):  
Kai Kruse ◽  
Noelia Díaz ◽  
Rocio Enriquez-Gasca ◽  
Xavier Gaume ◽  
Maria-Elena Torres-Padilla ◽  
...  
Keyword(s):  
Transposable Elements ◽  
Cell Stage ◽  
Genetic Components ◽  
Domain Boundaries ◽  
Stage Embryo ◽  
Chromatin Organisation ◽  
Early Mouse ◽  
Genome Activation

Transposable elements are abundant genetic components of eukaryotic genomes with important regulatory features affecting transcription, splicing, and recombination, among others. Here we demonstrate that the Murine Endogenous Retroviral Element (MuERV-L/MERVL) family of transposable elements drives the 3D reorganisation of the genome in the early mouse embryo. By generating Hi-C data in 2-cell-like cells, we show that MERLV elements promote the formation of insulating domain boundaries through-out the genome in vivo and in vitro. The formation of these boundaries is coupled to the upregulation of directional transcription from MERVL, which results in the activation of a subset of the gene expression programme of the 2-cell stage embryo. Domain boundaries in the 2-cell stage embryo are transient and can be remodelled without undergoing cell division. Remarkably, we find extensive inter-strain MERVL variation, suggesting multiple non-overlapping rounds of recent genome invasion and a high regulatory plasticity of genome organisation. Our results demonstrate that MERVL drive chromatin organisation during early embryonic development shedding light into how nuclear organisation emerges during zygotic genome activation in mammals.

Comparison of glucose metabolism in in vivo- and in vitro-matured tammar wallaby oocytes and its relationship to developmental potential following intracytoplasmic sperm injection

2004 ◽  
Vol 16 (6) ◽  
pp. 617 ◽  
Author(s):  
Genevieve M. Magarey ◽  
Karen E. Mate
Keyword(s):  
Glucose Metabolism ◽  
Intracytoplasmic Sperm Injection ◽  
Tammar Wallaby ◽  
Blastocyst Stage ◽  
In Vitro Fertilisation ◽  
Cell Stage ◽  
Developmental Potential ◽  
Developmental Capacity

Although marsupial oocytes undergo nuclear maturation in vitro, there is, at present, no indication of their developmental potential, largely owing to the lack of in vitro fertilisation and related technologies for marsupials. Glucose metabolism has proven a useful indicator of oocyte cytoplasmic maturation and developmental potential in several eutherian species. Therefore, the aims of the present study were to compare: (1) the rates of glycolysis and glucose oxidation in immature, in vitro-matured and in vivo-matured tammar wallaby oocytes; and (2) the metabolic rate of individual oocytes with their ability to form pronuclei after intracytoplasmic sperm injection. The rates of glycolysis measured in immature (2.18 pmol oocyte–1 h–1), in vitro-matured (0.93 pmol oocyte–1 h–1) and in vivo-matured tammar wallaby oocytes (0.54 pmol oocyte–1 h–1) were within a similar range to values obtained in eutherian species. However, unlike the trend observed in eutherian oocytes, the glycolytic rate was significantly higher in immature oocytes compared with either in vivo- or in vitro-matured oocytes (P < 0.001) and significantly higher in in vitro-matured oocytes compared with in vivo-matured oocytes (P < 0.001). No relationship was identified between glucose metabolism and the developmental capacity of oocytes after intracytoplasmic sperm injection when assessed after 17–19 h. Oocytes that became fertilised (two pronuclei) or activated (one or more pronucleus) were not distinguished from others by their metabolic rates. Longer culture after intracytoplasmic sperm injection (e.g. blastocyst stage) may show oocyte glucose metabolism to be predictive of developmental potential; however, culture to the single-cell stage did not reveal any significant differences in normally developing embryos.

Cyclin B1 levels in the porcine 4-cell stage embryo

Zygote ◽  
2002 ◽  
Vol 10 (1) ◽  
pp. 79-84 ◽  
Author(s):  
Aaron J. Bonk ◽  
Jon E. Anderson ◽  
Lalantha R. Abeydeera ◽  
Billy N. Day ◽  
Randall S. Prather
Keyword(s):  
Cyclin B1 ◽  
Cell Stage ◽  
Protein Levels ◽  
Cell Cleavage ◽  
Embryonic Genome ◽  
Stage Embryo ◽  
Polymerase Inhibitor ◽  
Genome Activation

The relative quantity of cyclin B1 was determined during the development of in vitro and in vivo derived porcine 4-cell embryos by western blotting and immunolocalised during the 4-cell stage. After cleavage to the 4-cell stage cyclin B1 localised to the cytoplasm at the 5, 10, 18 and 25 time points and localised to the nucleus 33 h post 4-cell cleavage (P4CC). The relative abundance of cyclin B1 was not significantly different in in vivo or in vitro derived 4-cell stage embryos cultured in the absence of the RNA polymerase inhibitor α-amanitin. Cyclin B1 protein was not detectable in embryos cultured in medium without α-amanitin for 5, 10, 18 or 25 h P4CC followed by culture in medium with α-amanitin to 33 P4CC. These results suggest that the maternal to zygotic transition of mRNA production that occurs at the 4-cell stage of the pig embryo does not result in an increase in cyclin B1 production. In addition, cyclin B1 protein levels remained constant in the absence of embryonic genome activation at the 4-cell stage.

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