Biology subjects

Fernandez, J. P.

Publications and source records attributed to Fernandez, J. P..

3 recordsLinked to original sources

bioRxiv2024

Optimizing Electroporation Parameters for Efficient Delivery of Large Molecules into Pig Zygotes Using Fluorescent Dextrans from 3 to 2000 kDA

Electroporation has revolutionized gene transfer and gene editing, enabling efficient delivery of molecules into embryos, with significant implications for developmental biology and biomedical research. This study aimed to optimize electroporation parameters for enhancing the delivery of large molecules into pig zygotes. We investigated the effects of fluorescence-coupled dextran reporters (FDs) of sizes ranging from 3 to 2000 kiloDalton (kDA) along with the impact of poring and transfer polarity settings during electroporation, on molecule permeability. Additionally, we assessed the influence of voltage and the number of poring pulses on the delivery of 2000 kDa FDs and examined the permeability of pre-IVF embryos and zona pellucida-weakened post-IVF embryos to this FD. Our findings highlighted size-dependent effects on FD uptake, with reversing poring polarity increasing the influx of small molecules (3 kDa FDs). The delivery of 2000 kDa FDs was not influenced by increased poring number but it was significantly influenced by voltage, reaching its optimum at 40 V. Electroporation in pre-IVF embryos did not show significant variation across different voltages. However, voltages higher than 20 V negatively affected blastocyst development rates. Zona-weakening did not improve permeability for the 2000 kDa FD. This study offers valuable insights into refining electroporation techniques for delivering large molecules into pig zygotes and highlights the relevance of commercial fluorescence-coupled dextrans as useful tools for exploring permeability dynamics in electroporated zygotes.

molecular biology↗

bioRxiv2018

Brd4 and P300 regulate zygotic genome activation through histone acetylation

The awakening of the zygote genome, signaling the transition from maternal transcriptional control to zygotic control, is a watershed in embryonic development, but the factors and mechanisms controlling this transition are still poorly understood. By combining CRISPR-Cas9-mediated live imaging of the first transcribed genes (miR-430), chromatin and transcription analysis during zebrafish embryogenesis, we observed that genome activation is gradual and stochastic, and the active state is inherited in daughter cells. We discovered that genome activation is regulated through both translation of maternal mRNAs and the effects of these factors on the chromatin. We show that chemical inhibition of H3K27Ac writer (P300) and reader (Brd4) block genome activation, while induction of a histone acetylation prematurely activates transcription, and restore genome activation in embryos where translation of maternal mRNAs is impaired, demonstrating that they are limiting factors for the activation of the genome. In contrast to current models, we do not observe triggering of genome activation by a reduction of the nuclear-cytoplasmic (N/C) ratio or slower cell division. We conclude that genome activation is controlled by a time-dependent mechanism involving the translation of maternal mRNAs and the regulation of histone acetylation through P300 and Brd4. This mechanism is critical to initiating zygotic development and developmental reprogramming.

developmental biology↗

bioRxiv2017

CRISPR-Cpf1 mediates efficient homology-directed repair and temperature-controlled genome editing

Cpf1 is a novel class of CRISPR-Cas DNA endonucleases, with a wide range of activity across different eukaryotic systems. Yet, the underlying determinants of this variability are poorly understood. Here, we demonstrate that LbCpf1, but not AsCpf1, ribonucleoprotein complexes allow efficient mutagenesis in zebrafish and Xenopus. We show that temperature modulates Cpf1 activity by controlling its ability to access genomic DNA. This effect is stronger on AsCpf1, explaining its lower efficiency in ectothermic organisms. We capitalize on this property to show that temporal control of the temperature allows post-translational modulation of Cpf1-mediated genome editing. Finally, we determine that LbCpf1 significantly increases homology-directed repair in zebrafish, improving current approaches for targeted DNA integration in the genome. Together, we provide a molecular understanding of Cpf1 activity in vivo and establish Cpf1 as an efficient and inducible genome engineering tool across ectothermic species.

genetics↗