Biology subjects

Hassel, P.

Publications and source records attributed to Hassel, 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↗

bioRxiv2021

Generation and first characterization of TRDC-Knockout pigs lacking γδ T cells

The TRDC-Locus encodes the T cell receptor delta constant region, one component of the {gamma}{delta} T cell receptor which is essential for development of {gamma}{delta} T cells. In contrast to peptide recognition by {beta} T cells, antigens activating {gamma}{delta} T cells are mostly MHC independent and not well characterized. Therefore, the function of {gamma}{delta} T cells and their contribution to protection against infections is still unclear. Higher numbers of circulating {gamma}{delta} T cells compared to mice, render the pig a suitable animal model to study {gamma}{delta} T cells. Knocking-out the porcine TRDC-locus by intracytoplasmic microinjection and somatic cell nuclear transfer resulted in healthy living {gamma}{delta} T cell deficient offspring. Flow cytometric analysis revealed that TRDC-KO pigs lack {gamma}{delta} T cells in peripheral blood mononuclear cells (PBMC) and spleen cells. The composition of the remaining leucocyte subpopulations was not affected by the depletion of {gamma}{delta} T cells. Genome-wide transcriptome analyses in PBMC revealed a pattern of changes reflecting the impairment of known or expected {gamma}{delta} T cell dependent pathways. Histopathology did not reveal developmental abnormalities of secondary lymphoid tissues. However, in a vaccination experiment the KO pigs stayed healthy but had a significantly lower neutralizing antibody titer as the syngenic controls.

immunology↗

bioRxiv2019

The knockout of the HMG-Domain of the porcine SRY-gene by CRISPR/Cas RNP microinjection causes sex reversal in gene-edited pigs

1The sex-determining region on the Y chromosome (SRY) is thought to be the central genetic element of male sex development. Mutations within the SRY gene are associated with a male-to-female sex reversal syndrome in humans and other mammalian species such as mice and rabbits. However, the underlying mechanisms are largely unknown. To understand the biological function of the SRY gene, a site-directed mutational analysis is required to investigate associated phenotypic changes at the molecular, cellular and morphological level. In our study, we successfully generated a knockout of the porcine SRY gene by microinjection of two clustered regularly interspaced short palindromic repeats (CRISPR) - associated protein - 9 nuclease (Cas9) ribonucleoprotein (RNP) complexes targeting the centrally located "high mobility group" (HMG) domain of the SRY gene. Mutations within this region resulted in the development of complete external and internal female genitalia in genetically male pigs. The internal female genitalia including uteri, ovaries, and oviducts, revealed substantial size differences in 9-months old SRY-knockout pigs compared to age-matched female wild type controls. In contrast, a deletion within the 5 flanking region of the HMG domain was not associated with sex reversal. Results of this study demonstrates for the first time the central role of the HMG domain of the SRY gene in male sex determination in pigs. Moreover, quantitative analysis by digital PCR revealed evidence for a duplication of the porcine SRY locus. Our results pave the way towards the generation of boars exclusively producing phenotypically female offspring to avoid surgical castration without anesthesia in piglets. Moreover, the study establishes a large animal model that is much more similar to humans in regard of physiology and anatomy and pivotal for longitudinal studies.

developmental biology↗