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"Xiang-xing, Zhu"
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CRISPR/Cas9-Mediated Generation of Guangxi Bama Minipigs Harboring Three Mutations in α-Synuclein Causing Parkinson’s Disease
Parkinson’s disease (PD) is a common, progressive neurodegenerative disorder characterized by classical motor dysfunction and is associated with α-synuclein-immunopositive pathology and the loss of dopaminergic neurons in the substantia nigra (SN). Several missense mutations in the α-synuclein gene
SCNA
have been identified as cause of inherited PD, providing a practical strategy to generate genetically modified animal models for PD research. Since minipigs share many physiological and anatomical similarities to humans, we proposed that genetically modified minipigs carrying PD-causing mutations can serve as an ideal model for PD research. In the present study, we attempted to model PD by generating Guangxi Bama minipigs with three PD-causing missense mutations (E46K, H50Q and G51D) in
SCNA
using CRISPR/Cas9-mediated gene editing combining with somatic cell nuclear transfer (SCNT) technique. We successfully generated a total of eight SCNT-derived Guangxi Bama minipigs with the desired heterozygous
SCNA
mutations integrated into genome, and we also confirmed by DNA sequencing that these minipigs expressed mutant α-synuclein at the transcription level. However, immunohistochemical analysis was not able to detect PD-specific pathological changes such as α-synuclein-immunopositive pathology and loss of SN dopaminergic neurons in the gene-edited minipigs at 3 months of age. In summary, we successfully generated Guangxi Bama minipigs harboring three PD-casusing mutations (E46K, H50Q and G51D) in
SCNA
. As they continue to develop, these gene editing minipigs need to be regularly teseted for the presence of PD-like pathological features in order to validate the use of this large-animal model in PD research.
Journal Article
Application of the modified cytosine base-editing in the cultured cells of bama minipig
Bama minipig is a unique miniature swine bred from China. Their favorable characteristics include delicious meat, strong adaptability, tolerance to rough feed, and high levels of stress tolerance. Unfavorable characteristics are their low lean meat percentage, high fat content, slow growth rate, and low feed conversion ratio. Genome-editing technology using CRISPR/Cas9 efficiently knocked out the myostatin gene (MSTN) that has a negative regulatory effect on muscle production, effectively promoting pig muscle growth and increasing lean meat percentage of the pigs. However, CRISPR/Cas9 genome editing technology is based on random mutations implemented by DNA double-strand breaks, which may trigger genomic off-target effects and chromosomal rearrangements. The application of CRISPR/Cas9 to improve economic traits in pigs has raised biosafety concerns. Base editor (BE) developed based on CRISPR/Cas9 such as cytosine base editor (CBE) effectively achieve targeted modification of a single base without relying on DNA double-strand breaks. Hence, the method has greater safety in the genetic improvement of pigs. The aim of the present study is to utilize a modified CBE to generate MSTN-knockout cells of Bama minipigs. Our results showed that the constructed “all-in-one”-modified CBE plasmid achieved directional conversion of a single C·G base pair to a T·A base pair of the MSTN target in Bama miniature pig fibroblast cells. We successfully constructed multiple single-cell colonies of Bama minipigs fibroblast cells carrying the MSTN premature termination and verified that there were no genomic off-target effects detected. This study provides a foundation for further application of somatic cell cloning to construct MSTN-edited Bama minipigs that carry only a single-base mutation and avoids biosafety risks to a large extent, thereby providing experience and a reference for the base editing of other genetic loci in Bama minipigs.
Journal Article
Efficient generation of GHR knockout Bama minipig fibroblast cells using CRISPR/Cas9-mediated gene editing
Dwarfism, also known as growth hormone deficiency (GHD), is a disease caused by genetic mutations that result in either a lack of growth hormone or insufficient secretion of growth hormone, resulting in a person's inability to grow normally. In the past, many studies focusing on GHD have made use of models of other diseases such as metabolic or infectious diseases. A viable GHD specific model system has not been used previously, thus limiting the interpretation of GHD results. The Bama minipig is unique to Guangxi province and has strong adaptability and disease resistance, and an incredibly short stature, which is especially important for the study of GHD. In addition, studies of GHR knockout Bama minipigs and GHR knockout Bama minipig fibroblast cells generated using CRISPR/Cas9 have not been previously reported. Therefore, the Bama minipig was selected as an animal model and as a tool for the study of GHD in this work. In this study, a Cas9 plasmid with sgRNA targeting the first exon of the GHR gene was transfected into Bama minipig kidney fibroblast cells to generate 22 GHR knockout Bama minipig kidney fibroblast cell lines (12 male monoclonal cells and 10 female monoclonal cells). After culture and identification, 11 of the 12 male clone cell lines showed double allele mutations, and the rate of positive alteration of GHR was 91.67%. Diallelic mutation of the target sequence occurred in 10 female clonal cell lines, with an effective positive mutation rate of 100%. Our experimental results not only showed that CRISPR/Cas9 could efficiently be used for gene editing in Bama minipig cells but also identified a highly efficient target site for the generation of a GHR knockout in other porcine models. Thus, the generation of GHR knockout male and female Bama fibroblast cells could lay a foundation for the birth of a future dwarfism model pig. We anticipate that the \"mini\" Bama minipig will be of improved use for biomedical and agricultural scientific research and for furthering our understanding of the genetic underpinnings of GHD.
Journal Article
Targeting autophagy in Alzheimer's disease: Animal models and mechanisms
Alzheimer's disease (AD) is an age-related progressive neurodegenerative disorder that leads to cognitive impairment and memory loss. Emerging evidence suggests that autophagy plays an important role in the pathogenesis of AD through the regulation of amyloid-beta (Aß) and tau metabolism, and that autophagy dysfunction exacerbates amyloidosis and tau pathology. Therefore, targeting autophagy may be an effective approach for the treatment of AD. Animal models are considered useful tools for investigating the pathogenic mechanisms and therapeutic strategies of diseases. This review aims to summarize the pathological alterations in autophagy in representative AD animal models and to present recent studies on newly discovered autophagy-stimulating interventions in animal AD models. Finally, the opportunities, difficulties, and future directions of autophagy targeting in AD therapy are discussed.
Journal Article
Adenine base-editing-mediated exon skipping induces gene knockout in cultured pig cells
Gene-knockout pigs have important applications in agriculture and medicine. Compared with CRISPR/Cas9, Adenine base editor (ABE) convert single A·T pairs to G·C pairs in the genome without generating DNA double-strand breaks, and this method has higher accuracy and biosafety in pig genetic modification. However, the application of ABE in pig gene knockout is limited by protospacer-adjacent motif sequences and the base-editing window. Alternative mRNA splicing is an important mechanism underlying the formation of proteins with diverse functions in eukaryotes. Spliceosome recognizes the conservative sequences of splice donors and acceptors in a precursor mRNA. Mutations in these conservative sequences induce exon skipping, leading to proteins with novel functions or to gene inactivation due to frameshift mutations. In this study, adenine base-editing-mediated exon skipping was used to expand the application of ABE in the generation of gene knockout pigs. We first constructed a modified “all-in-one” ABE vector suitable for porcine somatic cell transfection that contained an ABE for single-base editing and an sgRNA expression cassette. The “all-in-one” ABE vector induced efficient sgRNA-dependent A-to-G conversions in porcine cells during single base-editing of multiple endogenous gene loci. Subsequently, an ABE system was designed for single adenine editing of the conservative splice acceptor site (AG sequence at the 3′ end of the intron 5) and splice donor site (GT sequence at the 5′ end of the intron 6) in the porcine gene GHR; this method achieved highly efficient A-to-G conversion at the cellular level. Then, porcine single-cell colonies carrying a biallelic A-to-G conversion in the splice acceptor site in the intron 5 of GHR were generated. RT-PCR indicated exon 6 skipped at the mRNA level. Western blotting revealed GHR protein loss, and gene sequencing showed no sgRNA-dependent off-target effects. These results demonstrate accurate adenine base-editing-mediated exon skipping and gene knockout in porcine cells. This is the first proof-of-concept study of adenine base-editing-mediated exon skipping for gene regulation in pigs, and this work provides a new strategy for accurate and safe genetic modification of pigs for agricultural and medical applications.
Journal Article
Production of MSTN knockout porcine cells using adenine base-editing-mediated exon skipping
Gene-knockout pigs have important applications in agriculture and medicine. Compared with CRISPR/Cas9 and cytosine base editing (CBE) technologies, adenine base editing (ABE) shows better safety and accuracy in gene modification. However, because of the characteristics of gene sequences, the ABE system cannot be widely used in gene knockout. Alternative splicing of mRNA is an important biological mechanism in eukaryotes for the formation of proteins with different functional activities. The splicing apparatus recognizes conserved sequences of the 5′ end splice donor and 3′ end splice acceptor motifs of introns in pre-mRNA that can trigger exon skipping, leading to the production of new functional proteins, or causing gene inactivation through frameshift mutations. This study aimed to construct a
MSTN
knockout pig by inducing exon skipping with the aid of the ABE system to expand the application of the ABE system for the preparation of knockout pigs. In this study, first, we constructed ABEmaxAW and ABE8eV106W plasmid vectors and found that their editing efficiencies at the targets were at least sixfold and even 260-fold higher than that of ABEmaxAW by contrasting the editing efficiencies at the gene targets of endogenous
CD163
,
IGF2
, and
MSTN
in pigs. Subsequently, we used the ABE8eV106W system to realize adenine base (the base of the antisense strand is thymine) editing of the conserved splice donor sequence (5′-GT) of intron 2 of the porcine
MSTN
gene. A porcine single-cell clone carrying a homozygous mutation (5′-GC) in the conserved sequence (5′-GT) of the intron 2 splice donor of the
MSTN
gene was successfully generated after drug selection. Unfortunately, the
MSTN
gene was not expressed and, therefore, could not be characterized at this level. No detectable genomic off-target edits were identified by Sanger sequencing. In this study, we verified that the ABE8eV106W vector had higher editing efficiency and could expand the editing scope of ABE. Additionally, we successfully achieved the precise modification of the alternative splice acceptor of intron 2 of the porcine
MSTN
gene, which may provide a new strategy for gene knockout in pigs.
Journal Article
Successful cloning of an adult breeding boar from the novel Chinese Guike No. 1 swine specialized strain
Somatic cloning, also known as somatic cell nuclear transfer (SCNT), is a promising technology which has been expected to rapidly extend the population of elaborately selected breeding boars with superior production performance. Chinese Guike No. 1 pig breed is a novel swine specialized strain incorporated with the pedigree background of Duroc and Chinese Luchuan pig breeds, thus inherits an excellent production performance. The present study was conducted to establish somatic cloning procedures of adult breeding boars from the Chinese Guike No. 1 specialized strain. Ear skin fibroblasts were first isolated from a three-year-old Chinese Guike No. 1 breeding boar, and following that, used as donor cell to produce nuclear transfer embryos. Such cloned embryos showed full in vitro development and with the blastocyst formation rate of 18.4 % (37/201, three independent replicates). Finally, after transferring of 1187 nuclear transfer derived embryos to four surrogate recipients, six live piglets with normal health and development were produced. The overall cloning efficiency was 0.5 % and the clonal provenance of such SCNT derived piglets was confirmed by DNA microsatellite analysis. All of the cloned piglets were clinically healthy and had a normal weight at 1 month of age. Collectively, the first successful cloning of an adult Chinese Guike No. 1 breeding boar may lay the foundation for future improving the pig production industry.
Journal Article
Constitutive Relationship of New Steel 33Mn2V and Its Application in Piercing Process by FEM Simulation
Based on test data from the hot forge experiments on Gleeble 1500, a Kumar type constitutive equation for 33Mn2V steel is established. Applying this constitutive equation in commercial FEM software of MSC/SuperForm 2005, the piercing process of 33Mn2V steel in Mannesmann mill is then simulated. The modeling results visualized the dynamic evolution of equivalent stress, especially inside the workpieee. It is shown that the non-uniform distribu- tion of stress on the internal and external surface of the workpiece is a distinct characteristic of processing tube pierc- ing. The numerical model was verified by comparing the values of calculated force parameters of the piercing process with those measured in laboratory eonditions. And it shows that the Kumar-type constitutive relationship meets the practical needs.
Journal Article
Variability in phytoplankton biomass and effects of sea surface temperature based on satellite data from the Yellow Sea, China
A time series of satellite data on Chlorophyll-a concentration (Chl-a) that used ocean color was studied to determine mechanisms of phytoplankton variation in recent decade in the Yellow Sea, China during 2003-2015. The variability patterns on seasonal and inter-annual oscillation periods were confirmed using the Empirical Orthogonal Function (EOF), and Morlet wavelet transform analyses, respectively. The first EOF mode for Chl-a was dominated by obvious spring and fall blooms in a spatial pattern that was related to the strong mixing of the water masses from the Yellow Sea Cold Warm Mass (YSCWM) and the Yellow Sea Warm Current (YSWC) in winter. The second EOF mode for Chl-a showed an opposite spatial pattern between the northern and southern regions. The temporal coefficient showed differences in the timing of blooms. On an inter-annual scale, Chl-a indicated variation at periods of 2-4 years during 2003-2015. Chl-a showed a significantly negative correlation with the sea surface temperature (r = -0.21, p<0.01), with time lags of 4 months (Chl-a ahead). Chl-a was coupled with El Niño Southern Oscillation (ENSO) events, with a positive correlation (r = 0.46, p<0.01) at a lag of 3-5 months (ENSO ahead). The present study demonstrated that the variation in phytoplankton biomass was controlled primarily by water mass seasonally, and it was influenced by ENSO events on an inter-annual scale.
Journal Article