laser-assisted-lentiviral-gene-delivery-to-mouse-fertilized-eggs

Laser-assisted Lentiviral Gene Delivery to Mouse Fertilized Eggs

Lentiviruses are efficient vectors for gene delivery to mammalian cells. Following transduction, the lentiviral genome is stably incorporated into the ...

National Institute of Environmental Health Sciences

We have investigated the Rac-dependent mechanism of KCNH2 channel stimulation by thyroid hormone in a rat pituitary cell line, GH(4)C(1), with the patch-clamp technique. Here we present physiological evidence for the protein serine/threonine phosphatase, PP5, as an effector of Rac GTPase signaling. We also propose and test a specific molecular mechanism for PP5 stimulation by Rac-GTP. Inhibition of PP5 with the microbial toxin, okadaic acid, blocked channel stimulation by thyroid hormone and by Rac, but signaling was restored by expression of a toxin-insensitive mutant of PP5, Y451A, which we engineered. PP5 is unique among protein phosphatases in that it contains an N-terminal regulatory domain with three tetratricopeptide repeats (TPR) that inhibit its activity. Expression of the TPR domain coupled to GFP blocked channel stimulation by the thyroid hormone. We also show that the published structures of the PP5 TPR domain and the TPR domain of p67, the Rac-binding subunit of NADPH oxidase, superimpose over 92 alpha carbons. Mutation of the PP5 TPR domain at two predicted contact points with Rac-GTP prevents the TPR domain from functioning as a dominant negative and blocks the ability of Y451A to rescue signaling in the presence of okadaic acid. PP5 stimulation by Rac provides a unique molecular mechanism for the antagonism of Rho-dependent signaling through protein kinases in many cellular processes, including metastasis, immune cell chemotaxis, and neuronal development.

Rac GTPase signaling through the PP5 protein phosphatase.

The estrogen-related receptor α (ERRα) and the peroxisome proliferator-activated receptor γ (PPARγ) coactivator 1α (PGC-1α) play critical roles in the control of several physiological functions, including the regulation of genes involved in energy homeostasis. However, little is known about the ability of environmental chemicals to disrupt or modulate this important bioenergetics pathway in humans. The goal of this study was to develop a cell-based assay system with an intact PGC-1α/ERRα axis that could be used as a screening assay for detecting such chemicals. To this end, we successfully generated several stable cell lines expressing PGC-1α and showed that the reporter driven by the native ERRα hormone response unit (AAB-Luc) is active in these cell lines and that the activation is PGC-1α-dependent. Furthermore, we show that this activation can be blocked by the ERRα selective inverse agonist, XCT790. In addition, we find that genistein and bisphenol A further stimulate the reporter activity, while kaempferol has minimal effect. These cell lines will be useful for identifying environmental chemicals that modulate this important pathway. 

Development of a stable cell line with an intact PGC-1α/ERRα axis for screening environmental chemicals.

Lentiviruses are highly efficient vehicles for delivering genes into cells. They readily transduce primary and immortalized cells in vivo and in vitro. Genes delivered by lentiviruses are incorporated and replicated as part of their host genome and therefore offer a powerful tool for creation of stable cell lines and transgenic animals. However, the zona pellucida surrounding the fertilized eggs acts as a barrier and hinders lentiviral transduction of embryos. Here, we utilize a laser, typically used to perforate the zona pellucida for in vitro fertilization, to permeabilize the zona for lentiviral gene delivery. A single hole in the zona is sufficient for the lentivirus to gain access to fertilized eggs without the need for microinjection for en masse gene delivery. Embryos generated by this method elicit no damage and can develop to term for creation of transgenic animals.

Article	Year
Rac GTPase signaling through the PP5 protein phosphatase. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 16549782	2006
Development of a stable cell line with an intact PGC-1α/ERRα axis for screening environmental chemicals. Biochemical and biophysical research communications\| PubMed ID: 24457025	2014
En masse lentiviral gene delivery to mouse fertilized eggs via laser perforation of zona pellucida. Transgenic research\| PubMed ID: 29442214	2018

Charles Romeo

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