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In Vitro Cellular & Developmental... Jun 2024Genetic mosaicism, characterized by multiple genotypes within an individual, is considered an obstacle to CRISPR/Cas9 genome editing in animal models. Despite the...
Genetic mosaicism, characterized by multiple genotypes within an individual, is considered an obstacle to CRISPR/Cas9 genome editing in animal models. Despite the various strategies for minimizing mosaic mutations, no definitive methods exist to eliminate them. This study aimed to enhance gene editing efficiency in porcine zygotes using CRISPR/Cas9, which targets specific genes through centrifugation and zona pellucida removal before electroporation. Centrifugation at 2000 × g did not adversely affect blastocyst formation rates in zygotes electroporated with gRNA targeting the GGTA1 gene; instead, it led to increased total and monoallelic mutation rates compared with control zygotes without centrifugation. However, the groups had no significant differences in biallelic mutation rates. In zygotes electroporated with gRNA targeting the CMAH gene, centrifugation treatments exceeding 1000 × g significantly increased both biallelic mutation rates and mutation efficiency. The combination of centrifugation and zona pellucida removal did not have a detrimental effect on blastocyst formation rates. It led to a higher rate of double biallelic mutations in embryos targeting both GGTA1 and CMAH compared to embryos without centrifugation treatment. In summary, our results demonstrate that pre-electroporation treatments, including centrifugation and zona pellucida removal, positively influenced the reduction of mosaic mutations, with the effectiveness of centrifugation depending on the specific gRNA used.
PubMed: 38833208
DOI: 10.1007/s11626-024-00926-y -
Marine Biotechnology (New York, N.Y.) Jun 2024Specific cell depletion is a common means to study the physiological function of cell lineages and tissue regeneration. However, 100% depletion is difficult to achieve...
Specific cell depletion is a common means to study the physiological function of cell lineages and tissue regeneration. However, 100% depletion is difficult to achieve with existing cell depletion strategies. With the increasing maturity of CRISPR/Cas9 technology, it is increasingly used for the depletion of various cells. However, even with this technology, it is difficult to complete the depletion of specific gene knockout cells. For this reason, cell depletion with the use of repetitive sequences as the target of CRISPR/Cas9 was explored using zebrafish. All cells were used as the target cells for the first set of experiments. The results showed that injection of a mixture of DANA-gRNA and Cas9 mRNA into zygotes resulted in substantial cell apoptosis. Cells are almost invisible in the embryonic animal pole during the dome stage. The activities of the caspase-3 and caspase-9 proteins and the mRNA level of the P53 gene were significantly increased. Then, primordial germ cells (PGCs) in embryos were used as the target cells in subsequent experiments. To specifically knock out PGCs, we injected the mix of DANA-gRNA, pkop: Cas9 plasmid (the kop promotor allows Cas9 expression only in PGCs), and eGFP-nos3'UTR mRNA into zebrafish fertilized eggs. The results revealed that the activity of the caspase-3 protein was significantly increased, and the mRNA levels of P53, ku70, and ku80 were significantly upregulated, while the number of PGCs decreased gradually. Few PGCs labeled with GFP could be seen 20 h post-fertilization (hpf), and no PGCs could be seen at the germinal ridge 24 hpf. Therefore, the combination of CRISPR/Cas9 technology and repetitive sequences can achieve efficient cell depletion regardless of whether there is generalized expression or expression in specific cells. These results indicate that it is feasible to eliminate cells by using repeat sequences as CRISPR/Cas9 system target sites.
PubMed: 38833200
DOI: 10.1007/s10126-024-10328-6 -
The Journal of Clinical Endocrinology... Jun 2024The routine clinical practice is to prioritize the transfer of blastocysts derived from 2PN embryos if they are available. For women who only have blastocysts resulting...
CONTEXT
The routine clinical practice is to prioritize the transfer of blastocysts derived from 2PN embryos if they are available. For women who only have blastocysts resulting from 0PN and 1PN embryos, whether to transfer these embryos or discard them has been an ongoing debate over the years.
OBJECTIVE
To investigate the perinatal and obstetric outcomes following the transfer of vitrified-warmed single blastocysts derived from 0PN and 1PN zygotes.
DESIGN
Retrospective cohort study.
SETTING
University-affiliated IVF center.
PATIENT(S)
This study included singletons born to women who had undergone 0PN and 1PN vitrified-warmed single blastocyst transfers, compared to those resulting from 2PN vitrified-warmed single blastocyst transfers from 2012 to 2020.
INTERVENTIONS
None.
MAIN OUTCOME MEASURE(S)
Perinatal and obstetric outcomes.
RESULT(S)
A total of 7,284 women were included in the final analysis. Of these, 386, 316, and 6582 cycles resulted from 0PN-, 1PN-, and 2PN-derived blastocysts transfer, respectively. The rates of clinical pregnancy, miscarriage, and live birth were similar across the study cohorts in both unadjusted and adjusted analyses. When comparing the 0PN and 2PN groups, no differences were found in birth outcomes after adjusting for confounders. Similarly, maternal complications and mode of delivery were comparable between these two study cohorts. Birth parameters were also similar between the 1PN and 2PN blastocyst groups, except for more male births in the 1PN cohort. Furthermore, a comparison between the 1PN and 2PN groups did not reveal any significant differences in maternal outcomes.
CONCLUSION
The current study showed that the transfer of 0PN and 1PN blastocysts did not compromise reproductive outcomes or increase maternal and perinatal complications. This information is valuable for clinicians to counsel couples effectively and guide them in making informed decisions.
PubMed: 38832947
DOI: 10.1210/clinem/dgae375 -
Zygote (Cambridge, England) Jun 2024Spermatogenesis is a highly complex process through which mature sperms are produced, and it requires three important stages; mitosis, meiosis and sperm formation. The...
Spermatogenesis is a highly complex process through which mature sperms are produced, and it requires three important stages; mitosis, meiosis and sperm formation. The expression of genes regulated by transcription factors at specific stages exerts important regulatory effects on the development process of germ cells. Male mice with overexpressed programmed death ligand 1 (PD-L1) (B7 homolog1) in the testis have infertility and abnormal sperm development, thereby exhibiting severe malformation and sloughing throughout spermatid maturation and collapsed and disorganized seminiferous epithelium structure. Furthermore, PD-L1 overexpression causes overexpression of cysteine-rich secretory protein 1 (CRISP1) in the epididymis and adversely affects or precludes sperm energization, sperm-pellucida binding and sperm-oocyte fusion. These findings suggest that CRISP1 and PD-L1 can interact with each other to induce male infertility and germ-cell dissociation.
PubMed: 38828560
DOI: 10.1017/S0967199424000157 -
Zygote (Cambridge, England) Jun 2024The intra-ovarian presence of ootids, i.e. female gametes that have completed meiosis, is considered exceptional in the animal kingdom. The present study explores the...
The intra-ovarian presence of ootids, i.e. female gametes that have completed meiosis, is considered exceptional in the animal kingdom. The present study explores the first such case to be reported in a sea cucumber (Echinodermata: Holothuroidea). In the overwhelming majority of animals, including holothuroids, oocytes (i.e. immature female gametes) that are developing in the ovary undergo a primary arrest at the prophase stage of meiosis, which may last from days to decades. In free-spawning taxa, this arrest is normally lifted only during or shortly before transit in the gonoduct, when gamete release (spawning) is imminent. However, oocytes of the holothuroid were discovered to have resumed the second meiotic division including the completion of germinal vesicle breakdown and polar-body expulsion inside the ovary, effectively reaching the ootid stage concomitantly with ovulation (i.e. escape from follicle cells) prior to spawning. The potential drivers and significance of this exceptionally rare case of full intra-ovarian oogenic maturation are discussed.
PubMed: 38828553
DOI: 10.1017/S0967199424000170 -
Pharmacogenomics and Personalized... 2024The IQ motif and Sec7 domain ArfGEF 2 (), an X-linked gene that encodes the BRAG1 protein, is a guanine nucleotide exchange factor for the ADP ribosylation factor (ARF)...
BACKGROUND
The IQ motif and Sec7 domain ArfGEF 2 (), an X-linked gene that encodes the BRAG1 protein, is a guanine nucleotide exchange factor for the ADP ribosylation factor (ARF) protein family in the small guanosine triphosphate (GTP) binding protein. Mutations in this gene result in disorders such as intellectual disability (ID) and epilepsy. In this study, we analyze the clinical features of two patients with -mutation-related disease and discuss their possible pathogenesis.
METHODS
The two patients were diagnosed with ID and epilepsy. Genetic testing was performed using whole-exome sequencing, and the three-dimensional protein structure was analyzed. UCSC Genome Browser was used to analyze the conservation of in different species. We compared expression in the proband families with that in a control group, as well as the expression of the postsynaptic identity protein 95 (PSD-95), synapse-associated protein 97 (SAP97), ADP ribosylation factor 6 (ARF-6), and insulin receptor substrate 53kDa () genes interacting with .
RESULTS
We identified two semi-zygote mutations located in conserved positions in different species: an unreported mutation, C.3576C>A (p. Tyr1192*), and a known mutation, c.2983C>T (p. Arg995Trp). mutations resulted in significant changes in the predicted three-dimensional protein structure, while its expression in the two probands was significantly lower than that in the age-matched control group, and expression in proband 1 was lower than that in his family members. The expression levels of , and , which interact with , were also significantly different from those in the family members and age-matched healthy children.
CONCLUSION
The clinical phenotype resulting from mutations can be explained by the significant decrease in its expression, loss of function of the mutant protein, and change in the expression of related genes. Our results provide novel insights into the molecular phenotype conferred by the variants.
PubMed: 38827181
DOI: 10.2147/PGPM.S455840 -
BioRxiv : the Preprint Server For... May 2024The Maternal-to-Zygotic transition (MZT) is a reprograming process encompassing zygotic genome activation (ZGA) and the clearance of maternally-provided mRNAs. While...
The Maternal-to-Zygotic transition (MZT) is a reprograming process encompassing zygotic genome activation (ZGA) and the clearance of maternally-provided mRNAs. While some factors regulating MZT have been identified, there are thousands of maternal RNAs whose function has not been ascribed yet. Here, we have performed a proof-of-principle CRISPR-RfxCas13d maternal screening targeting mRNAs encoding protein kinases and phosphatases in zebrafish and identified Bckdk as a novel post-translational regulator of MZT. mRNA knockdown caused epiboly defects, ZGA deregulation, H3K27ac reduction and a partial impairment of miR-430 processing. Phospho-proteomic analysis revealed that Phf10/Baf45a, a chromatin remodeling factor, is less phosphorylated upon Bckdk depletion. Further, mRNA knockdown also altered ZGA and Phf10 constitutively phosphorylated rescued the developmental defects observed after mRNA depletion. Altogether, our results demonstrate the competence of CRISPR-RfxCas13d screenings to uncover new regulators of early vertebrate development and shed light on the post-translational control of MZT mediated by protein phosphorylation.
PubMed: 38826327
DOI: 10.1101/2024.05.22.595167 -
The Biological Bulletin Aug 2023AbstractWe describe the cloning and expression of a nonreceptor tyrosine kinase, (), a () gene, identified in a subtractive screen for maternal ascidian cDNAs in , an...
AbstractWe describe the cloning and expression of a nonreceptor tyrosine kinase, (), a () gene, identified in a subtractive screen for maternal ascidian cDNAs in , an ascidian species with a tadpole larva. The gene encodes a 4-kb mRNA expressed in gonads, eggs, and embryos in the tailed but is not detected in eggs or embryos of the closely related tailless species . There is a large insertion in in the genome, as shown by transcriptome and genome analyses, resulting in it becoming a pseudogene. The amino acid sequence encodes a nonreceptor tyrosine kinase with an N-terminal region containing two SH2 domains and five ankyrin repeats, similar to the gene found in other ascidians. Thus, the ascidian genes are members of the SHARK (Src-homology ankyrin-repeat containing tyrosine kinase) family of nonreceptor tyrosine kinases, which are found throughout invertebrates and missing from vertebrates. We show that is lacking the tyrosine kinase domain in the tailless , although the truncated mRNA is still expressed in transcriptome data. This maternal and zygotic tyrosine kinase is another described pseudogene from and appears not to be necessary for adult development.
Topics: Animals; Urochordata; Protein-Tyrosine Kinases; Amino Acid Sequence; Zygote; Pseudogenes; Phylogeny
PubMed: 38820291
DOI: 10.1086/730536 -
Cells & Development May 2024The oocyte expresses certain genes during folliculogenesis to regulate the acquisition of oocyte competence. Oocyte competence, or oocyte quality, is directly related to...
The oocyte expresses certain genes during folliculogenesis to regulate the acquisition of oocyte competence. Oocyte competence, or oocyte quality, is directly related to the ability of the oocyte to result in a successful pregnancy following fertilization. Presently, approximately 40 % of bovine embryos will develop to the blastocyst stage in vitro. Characterization of factors regulating these processes is crucial to improve the efficiency of bovine in vitro embryo production. We demonstrated that the secreted protein, agouti-signaling protein (ASIP) is highly abundant in the bovine oocyte and aimed to characterize its spatiotemporal expression profile in the ovary and throughout early embryonic development. In addition to oocyte expression, ASIP was detected in granulosa, cumulus, and theca cells isolated from antral follicles. Both gene expression data and immunofluorescent staining indicated ASIP declines with oocyte maturation which may indicate a potential role for ASIP in the attainment of oocyte competence. Microinjection of zygotes using small interfering RNA targeting ASIP led to a 16 % reduction in the rate of development to the blastocyst stage. Additionally, we examined potential ASIP signaling mechanisms through which ASIP may function to establish oocyte developmental competence. The expression of melanocortin receptor 3 and 4 and the coreceptor attractin was detected in the oocyte and follicular cells. The addition of cortisol during in vitro maturation was found to increase significantly oocyte ASIP levels. In conclusion, these results suggest a functional role for ASIP in promoting oocyte maturation and subsequent embryonic development, potentially through signaling mechanisms involving cortisol.
PubMed: 38815807
DOI: 10.1016/j.cdev.2024.203930 -
Cellular and Molecular Biology... May 2024Podocyte injury plays a vital role in focal segmental glomerulosclerosis (FSGS), and apoptosis is one of its mechanisms. The transient receptor potential channel 6...
Podocyte injury plays a vital role in focal segmental glomerulosclerosis (FSGS), and apoptosis is one of its mechanisms. The transient receptor potential channel 6 (TRPC6) is highly expressed in podocytes and mutations mediate podocyte injury. We found TRPC6 gene mutation (N110S) was a new mutation and pathogenic in the preliminary clinical work. The purpose of this study was to investigate the potential mechanism of mutation in TRPC6 (TRPC6-N110S) in the knock-in gene mouse model and in immortalized mouse podocytes (MPC5). Transmission electron microscopy was used to evaluate renal injury morphology. We measured 24-hour urinary albumin-to-creatinine ratios and major biochemical parameters such as serum albumin, urea nitrogen, and total cholesterol. The results of CCK-8 assay and apoptosis experiments showed that the TRPC6-N110S overexpression group had slower proliferative activity and increased apoptosis than the control group. FluO-3 assay revealed increased calcium influx in the TRPC6-N110S overexpression group. Podocin level was decreased in TRPC6-N110S group, while TRPC6 and desmin levels were increased in TRPC6-N110S group. The 24 h uACR at 6 weeks was significantly higher in the pure-zygotes group than in the WT and heterozygotes groups, and this difference was found at 8 and 10 weeks.TRPC6 levels showed no significant difference between homozygote and WT mice. Compared to homozygote group, expression of podocin and nephrin were increased in WT, but levels of desmin was decreased in WT. Our results suggest that this new mutation causes podocyte injury probably by enhancing calcium influx and podocyte apoptosis, accompanied by increased proteinuria and decreased expression of nephrin and podocin.
Topics: Podocytes; Animals; TRPC6 Cation Channel; Apoptosis; Mice; Gain of Function Mutation; Calcium; Glomerulosclerosis, Focal Segmental; Membrane Proteins; Desmin; Cell Proliferation; Intracellular Signaling Peptides and Proteins; TRPC Cation Channels; Male; Mice, Inbred C57BL
PubMed: 38814201
DOI: 10.14715/cmb/2024.70.5.42