Biology subjects

Gurumurthy, C. B.

Publications and source records attributed to Gurumurthy, C. B..

6 recordsLinked to original sources

bioRxiv2023

Delivering mRNAs to mouse tissues using the SEND system

mRNAs produced in a cell are almost always translated within the same cell. Some mRNAs are transported to other cells of the organism through processes involving membrane nanotubes or extracellular vesicles. A recent report describes a surprising new phenomenon of encapsulating mRNAs inside virus-like particles (VLPs) to deliver them to other cells in a process that was named SEND (Selective Endogenous eNcapsidation for cellular Delivery). Although the seminal work demonstrates the SEND process in cultured cells, it is unknown whether this phenomenon occurs in vivo. Here, we demonstrate the SEND process in living organisms using specially designed genetically engineered mouse models. Our proof of principle study lays a foundation for the SEND-VLP system to potentially be used as a gene therapy tool to deliver therapeutically important mRNAs to tissues.

genomics↗

bioRxiv2020

Slc26a9P2ACre, a new CRE driver to regulate gene expression in the otic placode lineage and other FGFR2b-dependent epithelia

Pan-otic CRE drivers enable gene regulation throughout the otic placode lineage, comprising the inner ear epithelium and neurons. However, intersection of extra-otic gene-of-interest expression with the CRE lineage can compromise viability and impede auditory analyses. Furthermore, extant pan-otic CREs recombine in auditory and vestibular brain nuclei, making it difficult to ascribe resulting phenotypes solely to the inner ear. We previously identified Slc26a9 as an otic placode-specific target of FGFR2b ligands, FGF3 and FGF10. We show here that Slc26a9 is otic-specific through E10.5, but not required for hearing. We targeted P2ACre to the Slc26a9 stop codon, generating Slc26a9P2ACre mice, and observed CRE activity throughout the otic epithelium and neurons, with little activity evident in the brain. Notably, recombination was detected in many FGFR2b ligand-dependent epithelia. We generated Fgf10 and Fgf8 conditional mutants, and activated an FGFR2b ligand trap from E17.5-P3. In contrast to analogous mice generated with other pan-otic CREs, these were viable. Auditory thresholds were elevated in mutants, and correlated with cochlear epithelial cell losses. Thus, Slc26a9P2ACre provides a useful complement to existing pan-otic CRE drivers, particularly for postnatal analyses. Summary statementWe describe a new pan-otic CRE driver, Slc26a9P2ACre, with little activity in the brain or middle ear, and demonstrate its utility by manipulating FGF signaling and assessing hearing loss.

developmental biology↗

bioRxiv2018

Human genome-edited hematopoietic stem cells phenotypically correct Mucopolysaccharidosis type I

Lysosomal enzyme deficiencies comprise a large group of genetic disorders that generally lack effective treatments. A potential treatment approach is to engineer the patients own hematopoietic system to express high levels of the deficient enzyme, thereby correcting the biochemical defect and halting disease progression. Here, we present an efficient ex vivo genome editing approach using CRISPR/Cas9 that targets the lysosomal enzyme iduronidase to the CCR5 safe harbor locus in human CD34+ hematopoietic stem and progenitor cells. The modified cells secrete supra-endogenous enzyme levels, maintain long-term repopulation and multi-lineage differentiation potential, and can correct biochemical and phenotypic abnormalities in an immunocompromised mouse model of Mucopolysaccharidosis type I. Our studies provide support for the development of human, genome-edited CD34+ hematopoietic stem and progenitor cells for the treatment of a multi-systemic lysosomal storage disorder. Our safe harbor approach constitutes a flexible platform for the expression of lysosomal enzymes, exemplifying a potential new paradigm for the treatment of these diseases.

genetics↗

bioRxiv2018

Human-like NSG mouse glycoproteins sialylation pattern changes the phenotype of human lymphocytes and sensitivity to HIV-1 infection

BackgroundThe use of immunodeficient mice transplanted with human hematopoietic stem cells is an accepted approach to study human-specific infectious diseases, like HIV-1, and to investigate multiple aspects of human immune system development. However, mouse and human are different in sialylation patterns of proteins due to evolutionary mutations of the CMP-N-acetylneuraminic acid hydroxylase (CMAH) gene that prevent formation of N-glycolylneuraminic acid from N-acetylneuraminic acid. How changes of mouse glycoproteins chemistry will affect phenotype and function of transplanted human hematopoietic stem cells and mature human immune cells in the course of HIV-1 infection is not known.\n\nResultsWe mutated mouse CMAH on the most widely human cells transplantation strain NOD/scid-IL2R{gamma} c-/- (NSG) mouse background using the CRISPR/Cas9 system. The new strain provides a better environment for human immune cells. Transplantation of human hematopoietic stem cells leads to broad B cells repertoire, higher sensitivity to HIV-1 infection, and enhanced proliferation of transplanted peripheral blood lymphocytes. The mice showed low effects on the clearance of human immunoglobulins and enhanced transduction efficiency of recombinant adeno-associated viral vector rAAV2/DJ8.\n\nConclusionNSG-cmah-/- mice expand the mouse models suitable for human cells transplantation and this new model has advantages in generating a human B cell repertoire. This strain is suitable to study different aspects of the human immune system development, might provide advantages in patient-derived tissue and cell transplantation, and could allow studies of viral vectors and infectious agents that are sensitive to human-like sialylation of mouse glycoproteins.

immunology↗

bioRxiv2018

Differential regulation of the ZsGreen1 reporter gene by the human cytomegalovirus promoter in 35 tissues of neonatal transgenic swine.

BackgroundThe advent of genetically engineered pig production has revealed a wide array of opportunities to enhance both biomedical and agricultural industries. One powerful method to develop these models is transgenesis; however, selection of a suitable promoter to drive transgene expression is critical. The cytomegalovirus (CMV) promoter is the most commonly used viral promoter as it robustly drives transgene expression in a ubiquitous nature. However, recent reports suggest that the level of CMV promoter activity is tissue-dependent in the pig. Therefore, the objective of this study was to quantify the activity of the CMV promoter in a wide range of porcine tissues. Swine harboring a CMV-ZsGreen1 transgene with a single integration site were utilized for this study. Thirty five tissue samples were collected from neonatal hemizygous (n = 3) and homozygous (n = 3) transgenic piglets and analyzed for ZsGreen1 abundance via immunoblot.\n\nResultsZsGreen1 was detected in all tissues examined; however, quantification revealed that ZsGreen1 protein levels were tissue-specific. Within organs of the digestive system, for example, ZsGreen1 was most abundant in the salivary gland, moderately produced in the esophagus and levels were lowest in the stomach. Interestingly, abundance of ZsGreen1 also differed within organ. For instance, levels were highest in the right ventricle compared with other chambers of the heart. There was no effect of transgene dose as ZsGreen1 expression patterns were similar between homozygous and hemizygous piglets.\n\nConclusionsUltimately, these results elucidate the tissue-specific activity of the CMV promoter in the neonatal pig. Moreover, this model can serve as a useful tool for research applications requiring reporter gene activity in mammalian organs.

genomics↗

bioRxiv2017

An Efficient CRISPR Protocol For Generating Conditional And Knock-In Mice Using Long Single-Stranded DNA Donors

The CRISPR/Cas9 tool can easily generate knockout mouse models by disrupting the gene sequence, but its efficiency for creating models that require either insertion of exogenous DNA (knock-in) or replacement of genomic segments is very poor. The majority of mouse models used in research are knock-in (reporters or recombinases) or gene-replacement (for example, conditional knockout alleles containing LoxP sites flanked exons). A few methods for creating such models are reported using double-stranded DNA as donors, but their efficiency is typically 1-10% and therefore not suitable for routine use. We recently demonstrated that long single-stranded DNAs serve as very efficient donors, both for insertion and for gene replacement. We call this method Easi-CRISPR (efficient additions with ssDNA inserts-CRISPR), a highly efficient technology (typically 25%-50%, and up to 100% in some cases), one that has worked at over a dozen loci thus far. Here, we provide detailed protocols for Easi-CRISPR.

genomics↗