Biology subjects

Rivera-Perez, J. A.

Publications and source records attributed to Rivera-Perez, J. A..

4 recordsLinked to original sources

bioRxiv2024

Early zygotic engineering promotes targeted large transgene integration and direct production of fully transgenic animals

Genetic engineering has become increasingly feasible with the advent of site-specific nucleases. This technology is highly effective for the generation of small mutations, however targeted insertion of large sequences to generate transgenic animals remains challenging, time consuming, and laborious. Following several failed attempts using the same reagents, we identified a protocol that achieved very high targeted integration of a 3.2 kb transgene into a single locus of a humanized mouse model of Down syndrome. Moreover, we demonstrate in multiple ways that this procedure directly generates numerous non-mosaic founder animals, which bred true to generate all transgenic progeny. In vitro fertilization of oocytes was followed by AAV-mediated delivery of donor sequence and electroporation of Cas9 ribonucleoprotein, all parameters designed to promote early zygotic engineering (EZE). This strategy can obviate the need to breed mosaic animals for germline transmission and can enable engineering of difficult to breed animals. While efforts to improve genome engineering focus on reagents and delivery techniques, findings here suggest a narrow window of time in the early fertilized oocyte, likely before genome replication, is key to achieve both high integration efficiency and one-step generation of non-mosaic, engineered mice.

developmental biology↗

bioRxiv2022

Sfrp2 is a multifunctional regulator of rodent color patterns

Animal pigment patterns are excellent models to elucidate mechanisms of biological organization. Although theoretical simulations, such as Turing reaction-diffusion systems, recapitulate many animal patterns, they are insufficient to account for those showing a high degree of spatial organization and reproducibility. Here, we compare the coats of the African striped mouse (Rhabdomys pumilio) and the laboratory mouse (Mus musculus) to study the molecular mechanisms controlling stripe pattern formation. By combining transcriptomics, mathematical modeling, and mouse transgenics, we show that Sfrp2 regulates the distribution of hair follicles and establishes an embryonic prepattern that foreshadows pigment stripes. Moreover, by developing and employing in vivo gene editing experiments in striped mice, we find that Sfrp2 knockout is sufficient to alter the stripe pattern. Strikingly, mutants also exhibit changes in coat color, revealing an additional function of Sfrp2 in regulating hair color. Thus, a single factor controls coat pattern formation by acting both as an orienting signaling mechanism and a modulator of pigmentation. By uncovering a multifunctional regulator of stripe formation, our work provides insights into the mechanisms by which spatial patterns are established in developing embryos and the molecular basis of phenotypic novelty.

developmental biology↗

bioRxiv2020

Implantation and Gastrulation Abnormalities Characterize Early Embryonic Lethal Mouse Lines

The period of development between the zygote and embryonic day 9.5 in mice includes multiple developmental milestones essential for embryogenesis. The preeminence of this period of development has been illustrated in loss of function studies conducted by the International Mouse Phenotyping Consortium (IMPC) which have shown that close to one third of all mouse genes are essential for survival to weaning age and a significant number of mutations cause embryo lethality before E9.5. Here we report a systematic analysis of 21 pre-E9.5 lethal lines generated by the IMPC. Analysis of pre- and post-implantation embryos revealed that the majority of the lines exhibit mutant phenotypes that fall within a window of development between implantation and gastrulation with few pre-implantation and no post-gastrulation phenotypes. Our study provides multiple genetic inroads into the molecular mechanisms that control early mammalian development and the etiology of human disease, in particular, the genetic bases of infertility and pregnancy loss. We propose a strategy for an efficient assessment of early embryonic lethal mutations that can be used to assign phenotypes to developmental milestones and outline the time of lethality.

developmental biology↗

bioRxiv2019

CK2-dependent phosphorylation of the Brg1 chromatin remodeling enzyme occurs during mitosis

Brg1 (Brahma related gene 1) is one of two mutually exclusive ATPases that can act as the catalytic subunit of mammalian SWI/SNF chromatin remodeling enzymes that facilitate utilization of the DNA in eukaryotic cells. Brg1 is a phospho-protein and its activity is regulated by specific kinases and phosphatases. Previously, we showed that Brg1 interacts with and is phosphorylated by casein kinase 2 (CK2) in a manner that regulates myoblast proliferation. Here we demonstrate that the Brg1-CK2 interaction occurred during mitosis in embryonic somites and in primary myoblasts derived from satellite cells isolated from muscle tissue. The interaction of CK2 activity with Brg1 and the incorporation of a number of other subunits into the mSWI/SNF enzyme complex were independent of CK2 enzymatic activity. CK2-mediated hyperphosphorylation of Brg1 was observed in mitotic cells derived from multiple cell types and organisms, suggesting functional conservation across tissues and species. The mitotically hyperphosphorylated form of Brg1 was localized with soluble chromatin, demonstrating that CK2-mediated phosphorylation of Brg1 is associated with specific partitioning of Brg1 within sub-cellular compartments. Thus CK2 acts a mitotic kinase that regulates Brg1 phosphorylation and sub-cellular localization.\n\nHIGHLIGHTSO_LIInteractions between CK2 and the Brg1 chromatin remodeling enzyme occur during mitosis\nC_LIO_LICK2-Brg1 interactions are independent of CK2 catalytic activity\nC_LIO_LICK2-mediated phosphorylation of Brg1 is a mitotic event\nC_LIO_LICK2-mediated phosphorylation of Brg1 is conserved across mammalian cell types\nC_LIO_LIThe mitotically hyperphosphorylated form of Brg1 is localized with soluble chromatin\nC_LI

cell biology↗