CRISPR/Cas9 is becoming an increasingly important tool to functionally annotate genomes. However, because genome-wide CRISPR libraries are mostly constructed in lentiviral vectors, in vivo ...applications are severely limited as a result of difficulties in delivery. Here, we examined the piggyBac (PB) transposon as an alternative vehicle to deliver a guide RNA (gRNA) library for in vivo screening. Although tumor induction has previously been achieved in mice by targeting cancer genes with the CRISPR/Cas9 system, in vivo genome-scale screening has not been reported. With our PB-CRISPR libraries, we conducted an in vivo genome-wide screen in mice and identified genes mediating liver tumorigenesis, including known and unknown tumor suppressor genes (TSGs). Our results demonstrate that PB can be a simple and nonviral choice for efficient in vivo delivery of CRISPR libraries.
Targeted mutagenesis in model organisms is key for gene functional annotation and biomedical research. Despite technological advances in gene editing by the CRISPR-Cas9 systems, rapid and efficient ...introduction of site-directed mutations remains a challenge in large animal models. Here, we developed a robust and flexible insertional mutagenesis strategy, homology-independent targeted trapping (HIT-trapping), which is generic and can efficiently target-trap an endogenous gene of interest independent of homology arm and embryonic stem cells. Further optimization and equipping the HIT-trap donor with a site-specific DNA inversion mechanism enabled one-step generation of reversible and conditional alleles in a single experiment. As a proof of concept, we successfully created mutant alleles for 21 disease-related genes in primary porcine fibroblasts with an average knock-in frequency of 53.2%, a great improvement over previous approaches. The versatile HIT-trapping strategy presented here is expected to simplify the targeted generation of mutant alleles and facilitate large-scale mutagenesis in large mammals such as pigs.
To date no authentic embryonic stem cell (ESC) line or germline-competent-induced pluripotent stem cell (iPSC) line has been established for large animals. Despite this fact, there is an impression ...in the field that large animal ESCs or iPSCs are as good as mouse counterparts. Clarification of this issue is important for a healthy advancement of the stem cell field. Elucidation of the causes of this failure in obtaining high quality iPSCs/ESCs may offer essential clues for eventual establishment of authentic ESCs for large animals including humans. To this end, we first generated porcine iPSCs using nonintegrating replicating episomal plasmids. Although these porcine iPSCs met most pluripotency criteria, they could neither generate cloned piglets through nuclear transfer, nor contribute to later stage chimeras through morula injections or aggregations. We found that the reprogramming genes in iPSCs could not be removed even under negative selection, indicating they are required to maintain self-renewal. The persistent expression of these genes in porcine iPSCs in turn caused differentiation defects in vivo. Therefore, incomplete reprogramming manifested by a reliance on sustained expression of exogenous-reprogramming factors appears to be the main reason for the inability of porcine iPSCs to form iPSC-derived piglets.
Although the role of asialoglycoprotein receptor 1 (ASGR1) in lowering lipid levels is well established, recent studies indicate that ASGR1 inhibition can cause unexpected liver damage in pigs, ...raising a serious issue about whether ASGR1 can be a good target for treating ASCVD. Here, we utilized the CRISPR-Cas9 system to regenerate ASGR1-knockout pigs, who displayed decreased lipid profiles without observable liver damage. This was confirmed by the lower levels of serum ALT and AST, reduced expression of inflammation markers, and normal histological morphology. Also, we implemented immunoprecipitation combined with mass spectrometry (IP-MS) and discovered that paraoxonase-2 (PON2) can interact with and significantly degrade ASGR1 in a dose-dependent manner. This degradation reduced lipid levels in mice, accompanied by little inflammation. Our study highlights the effectiveness and safety of degrading ASGR1 to reduce lipid levels in pigs and provides a potential inhibitor of ASGR1.
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•ASGR1+/− pigs present a low lipid profile with no obvious inflammation•PON2 binds ASGR1 and induces its degradation•Overexpression of PON2 decreases ASGR1 and blood cholesterol levels in mice
Human metabolism; Genetics
CMF+AE for EU Waste Water Du, Xuguang; Liu, Xuejun
Energy procedia,
2013, 2013-00-00, Letnik:
39
Journal Article
Recenzirano
Odprti dostop
A great deal of research has been directed towards the problem of the treatment of waste water contaminated by uranium, and lots of processes available in nuclear facility. However, the key problem ...is that the existing processes are mainly applied for depleted uranium (DU), but not for enriched uranium (EU). So, it's imperative to develop a process for EU waste water treatment which comes from nuclear facility decommission or others.
Consequently, the new process coagulation micro-filtration (CMF) combined with anion exchange (AE) was established to deal with the EU waste water. The experiments was divided into three parts CMF, AE and CMF+AE, respectively. The experiment results revealed that CMF is capable to deal with the waste water(the concentration of uranium in original waste water was 4.29mg/L) at the pH 6.0-7.0, and its decontamination factor (DF) reached 103;AE is good at treating the original waste water when the concentration of which is not more than 500g/L, meanwhile, the residence time is not less than 10minutes, therefore, the treated water by AE meets the discharge standard; The DF number of the combined CMF + AE process might get 104.
According to our research, the CMF + AE process provides a new choice for EU waste water treatment.
Dosage compensation, which is achieved by X-chromosome inactivation (XCI) in female mammals, ensures balanced X-linked gene expression levels between the sexes. Although eutherian mammals commonly ...display random XCI in embryonic and adult tissues, imprinted XCI has also been identified in extraembryonic tissues of mouse, rat, and cow. Little is known about XCI in pigs. Here, we sequenced the porcine
XIST
gene and identified an insertion/deletion mutation between Asian- and Western-origin pig breeds. Allele-specific analysis revealed biallelic
XIST
expression in porcine ICSI blastocysts. To investigate the XCI pattern in porcine placentas, we performed allele-specific RNA sequencing analysis on individuals from reciprocal crosses between Duroc and Rongchang pigs. Our results were the first to reveal that random XCI occurs in the placentas of pigs. Next, we investigated the H3K27me3 histone pattern in porcine blastocysts, showing that only 17–31.8% cells have attained XCI. The hypomethylation status of an important
XIST
DMR (differentially methylated region) in gametes and early embryos demonstrated that no methylation is pre-deposited on
XIST
in pigs. Our findings reveal that the XCI regulation mechanism in pigs is different from that in mice and highlight the importance of further study of the mechanisms regulating XCI during early porcine embryo development.
Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne bunyavirus causing a high fatality rate of up to 30%. To date, the receptor mediating SFTSV entry remained ...uncharacterized, hindering the understanding of disease pathogenesis. Here, C-C motif chemokine receptor 2 (CCR2) was identified as a host receptor for SFTSV based on a genome-wide CRISPR-Cas9 screen. Knockout of CCR2 substantially reduced viral binding and infection. CCR2 enhanced SFTSV binding through direct binding to SFTSV glycoprotein N (Gn), which is mediated by its N-terminal extracellular domain. Depletion of CCR2 in C57BL/6J mouse model attenuated SFTSV replication and pathogenesis. The peripheral blood primary monocytes from elderly individuals or subjects with underlying diabetes mellitus showed higher CCR2 surface expression and supported stronger binding and replication of SFTSV. Together, these data indicate that CCR2 is a host entry receptor for SFTSV infection and a novel target for developing anti-SFTSV therapeutics.
Efficient immune responses rely on the proper differentiation of CD8
T cells into effector and memory cells. Here, we show a critical requirement of N
-Methyladenosine (m
A) methyltransferase Mettl3 ...during CD8
T cell responses upon acute viral infection. Conditional deletion of Mettl3 in CD8
T cells impairs effector expansion and terminal differentiation in an m
A-dependent manner, subsequently affecting memory formation and the secondary response of CD8
T cells. Our combined RNA-seq and m
A-miCLIP-seq analyses reveal that Mettl3 deficiency broadly impacts the expression of cell cycle and transcriptional regulators. Remarkably, Mettl3 binds to the Tbx21 transcript and stabilizes it, promoting effector differentiation of CD8
T cells. Moreover, ectopic expression of T-bet partially restores the defects in CD8
T cell differentiation in the absence of Mettl3. Thus, our study highlights the role of Mettl3 in regulating multiple target genes in an m
A-dependent manner and underscores the importance of m
A modification during CD8
T cell response.
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