Precise and efficient genome modifications provide powerful tools for biological studies. Previous CRISPR gene knockout methods in cell lines have relied on frameshifts caused by stochastic ...insertion/deletion in all alleles. However, this method is inefficient for genes with high copy number due to polyploidy or gene amplification because frameshifts in all alleles can be difficult to generate and detect. Here we describe a homology-directed insertion method to knockout genes in the polyploid Drosophila S2R+ cell line. This protocol allows generation of homozygous mutant cell lines using an insertion cassette which autocatalytically generates insertion mutations in all alleles. Knockout cells generated using this method can be directly identified by PCR without a need for DNA sequencing. This protocol takes 2-3 months and can be applied to other polyploid cell lines or high-copy-number genes.
Loss-of-function and gain-of-function genetic perturbations provide valuable insights into gene function. In
cells, while genome-wide loss-of-function screens have been extensively used to reveal ...mechanisms of a variety of biological processes, approaches for performing genome-wide gain-of-function screens are still lacking. Here, we describe a pooled CRISPR activation (CRISPRa) screening platform in
cells and apply this method to both focused and genome-wide screens to identify rapamycin resistance genes. The screens identified three genes as novel rapamycin resistance genes: a member of the SLC16 family of monocarboxylate transporters (
), a member of the lipocalin protein family (
), and a zinc finger C2H2 transcription factor (
). Mechanistically, we demonstrate that
overexpression activates the RTK-Akt-mTOR signaling pathway and that activation of insulin receptor (InR) by
requires cholesterol and clathrin-coated pits at the cell membrane. This study establishes a novel platform for functional genetic studies in
cells.
Uncovering the complexity of systems in non-model organisms is critical for understanding arthropod immunology. Prior efforts have mostly focused on Dipteran insects, which only account for a subset ...of existing arthropod species in nature. Here we use and develop advanced techniques to describe immune cells (hemocytes) from the clinically relevant tick Ixodes scapularis at a single-cell resolution. We observe molecular alterations in hemocytes upon feeding and infection with either the Lyme disease spirochete Borrelia burgdorferi or the rickettsial agent Anaplasma phagocytophilum. We reveal hemocyte clusters exhibiting defined signatures related to immunity, metabolism, and proliferation. Depletion of phagocytic hemocytes affects hemocytin and astakine levels, two I. scapularis hemocyte markers, impacting blood-feeding, molting behavior, and bacterial acquisition. Mechanistically, astakine alters hemocyte proliferation, whereas hemocytin affects the c-Jun N-terminal kinase (JNK) signaling pathway in I. scapularis. Altogether, we discover a role for tick hemocytes in immunophysiology and provide a valuable resource for comparative biology in arthropods.
Abnormal oocytes are one of the major causes of reproductive failure, and also limit the efficiency of assisted reproduction 1. The generation of functional oocytes either by differentiation of ...pluripotent stem cells 2 or in vitro culturing of the embryonic genital ridges 3 has not yet been achieved. Recently, we and other groups have reported that live fertile mice could be successfully produced by injecting androgenetic haploid embryonic stem （ahES） cells that carry some paternal imprints into oocytes in place of sperm 4, 5.
Nutrient deprivation induces autophagy through inhibiting TORC1 activity. We describe a novel mechanism in Drosophila by which TORC1 regulates RNA processing of Atg transcripts and alters ATG protein ...levels and activities via the cleavage and polyadenylation (CPA) complex. We show that TORC1 signaling inhibits CDK8 and DOA kinases, which directly phosphorylate CPSF6, a component of the CPA complex. These phosphorylation events regulate CPSF6 localization, RNA binding, and starvation-induced alternative RNA processing of transcripts involved in autophagy, nutrient, and energy metabolism, thereby controlling autophagosome formation and metabolism. Similarly, we find that mammalian CDK8 and CLK2, a DOA ortholog, phosphorylate CPSF6 to regulate autophagy and metabolic changes upon starvation, revealing an evolutionarily conserved mechanism linking TORC1 signaling with RNA processing, autophagy, and metabolism.
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•TORC1 negatively inhibits CDK8 and DOA kinases•CDK8 and DOA phosphorylate CPSF6 to induce alternative RNA processing•Depletion of CPSF6 impairs autophagy and metabolic changes during starvation•The CDK8-CLK2/DOA-CPSF6 axis is conserved in mammals
Tang et al. investigate the mechanisms of how TORC1 regulates autophagy and cell metabolism. They demonstrate that CDK8 and DOA, two kinases downstream of TORC1 signaling, directly phosphorylate CPSF6 to regulate alternative mRNA polyadenylation and splicing and mediate TORC1-dependent physiological functions.
How do different cell types acquire their specific identities and functions is a fundamental question of biology. Previously significant efforts have been devoted to search for cell‐type‐specifically ...expressed genes, especially transcription factors, yet how do ubiquitously expressed genes participate in the formation or maintenance of cell‐type‐specific features remains largely unknown. Here, we have identified 110 mouse embryonic stem cell (mESC) specifically expressed transcripts with cell‐stage‐specific alternative transcription start sites (SATS isoforms) from 104 ubiquitously expressed genes, majority of which have active epigenetic modification‐ or stem cell‐related functions. These SATS isoforms are specifically expressed in mESCs, and tend to be transcriptionally regulated by key pluripotency factors through direct promoter binding. Knocking down the SATS isoforms of Nmnat2 or Usp7 leads to differentiation‐related phenotype in mESCs. These results demonstrate that cell‐type‐specific transcription factors are capable to produce cell‐type‐specific transcripts with alternative transcription start sites from ubiquitously expressed genes, which confer ubiquitously expressed genes novel functions involved in the establishment or maintenance of cell‐type‐specific features.
Synopsis
This study shows that ubiquitously expressed genes can be involved in the establishment or maintenance of cell‐type‐specific features via producing cell‐type‐specific transcripts with alternative transcription start sites, some of which are regulated by cell‐type‐specific transcription factors.
Some ubiquitously expressed genes produce transcripts with stage‐specific alternative transcription start sites (SATS) in mESCs.
Key pluripotency factors regulate the expression of some SATS isoforms.
In mESCs, SATS isoforms are required for the maintenance of pluripotency.
This study shows that ubiquitously expressed genes can be involved in the establishment or maintenance of cell‐type‐specific features via producing cell‐type‐specific transcripts with alternative transcription start sites, some of which are regulated by cell‐type‐specific transcription factors.
The pluripotency of embryonic stem cells (ESCs) is controlled by a multilayer regulatory network, of which the key factors include core pluripotency genes Oct4, Sox2 and Nanog, and multiple microRNAs ...(miRNAs). Recently, long noncoding RNAs (lncRNAs) have been discovered as a class of new regulators for ESCs, and some lncRNAs could function as competing endogenous RNAs (ceRNAs) to regulate mRNAs by competitively binding to miRNAs. Here, we identify mmu-miR-139-5p as a new regulator for Nanog by targeting Nanog 3′ untranslated region (UTR) to repress Nanog expression in mouse ESCs and embryos. Such regulation could be released by an ESC-specifically expressed ceRNA named lnc-NAP. The expression of lnc-NAP is activated by OCT4, SOX2, as well as NANOG through promoter binding. Downregulation of lnc-NAP reduces Nanog abundance, which leads to decreased pluripotency of mouse ESCs and embryonic lethality. These results reveal lnc-NAP as a new regulator for Nanog in mouse ESCs, and uncover a feed-forward regulatory loop of Nanog through the participation of lnc-NAP.
Embryonic stem cells (ESCs) can grow infinitely and give rise to all types of cells in human body, thus of tremendous therapeutic potentials for a variety of diseases, such as Parkinson's disease, ...spinal cord injury, and diabetes. Moreover, the combination of gene modification and directed differentiation of ESCs provides perfect tool for disease modelling and drug discovery. However, tissue rejection following ESCs derivatives transplantation greatly hinders its application. Under such circumstances, the idea of "therapeutic cloning" was proposed, indicating the generation of ESCs from SCNT embryos for therapeutic purpose. Mouse nuclear transfer embryonic stem cells (NT-ESCs) were first established in 2000, and then proved to be able to differentiate either in vivo or in vitro, and give rise to individual tissues through germ line transmission or tetraploid complementation. Fully reprogrammed NT-ESCs are indistinguishable from ESCs derived from fertilized eggs functionally and substantially. What is more, by deriving NT-ESCs from patient cells, the problem of immune rejection may be avoided. However, the derivation of human NT-ESCs goes with the destruction of clone embryos, leading to fierce ethical disputes. There has not been report of successful establishment of human NT-ES cells so far, and the limited resource of human eggs used for nuclear transfer cumbers the future application of NT-ESCs. In this chapter, we will introduce therapeutic cloning in two aspects as SCNT and NT-ESCs, and the history, nowadays status and prospects of them will be reviewed.
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