Insertion Of Fragment Research Articles

CRISPR/Cas-mediated DNA base editing technology and its application in biomedicine and agriculture

In recent years, the genome editing technologies based on the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) have developed rapidly. The system can use homologous directed recombination (HDR) to achieve precise editing that it medicated, but the efficiency is extremely low, which limits its application in agriculture and biomedical fields. As an emerging genome editing technology, the CRISPR/Cas-mediated DNA base editing technologies can achieve targeted mutations of bases without generating double-strand breaks, and has higher editing efficiency and specificity compared with CRISPR/Cas-mediated HDR editing. At present, cytidine base editors (CBEs) that can mutate C to T, adenine base editors (ABEs) that can mutate A to G, and prime editors (PEs) that enable arbitrary base conversion and precise insertion and deletion of small fragments, have been developed. In addition, glycosylase base editors (GBEs) capable of transitioning from C to G and double base editors capable of editing both A and C simultaneously, have been developed. This review summarizes the development, advances, advantages and limitations of several DNA base editors. The successful applications of DNA base editing technology in biomedicine and agriculture, together with the prospects for further optimization and selection of DNA base editors, are discussed.

Comparative genome analysis of Clostridium beijerinckii strains isolated from pit mud of Chinese strong flavor baijiu ecosystem.

Clostridium beijerinckii is a well-known anaerobic solventogenic bacterium which inhabits a wide range of different niches. Previously, we isolated five butyrate-producing C. beijerinckii strains from pit mud (PM) of strong-flavor baijiu (SFB) ecosystems. Genome annotation of the five strains showed that they could assimilate various carbon sources as well as ammonium to produce acetate, butyrate, lactate, hydrogen, and esters but did not produce the undesirable flavors isopropanol and acetone, making them useful for further exploration in SFB production. Our analysis of the genomes of an additional 233 C. beijerinckii strains revealed an open pangenome based on current sampling and will likely change with additional genomes. The core genome, accessory genome, and strain-specific genes comprised 1567, 8851, and 2154 genes, respectively. A total of 298 genes were found only in the five C. beijerinckii strains from PM, among which only 77 genes were assigned to Clusters of Orthologous Genes categories. In addition, 15 transposase and 12 phage integrase families were found in all five C. beijerinckii strains from PM. Between 18 and 21 genome islands were predicted for the five C. beijerinckii genomes. The existence of a large number of mobile genetic elements indicated that the genomes of the five C. beijerinckii strains evolved with the loss or insertion of DNA fragments in the PM of SFB ecosystems. This study presents a genomic framework of C. beijerinckii strains from PM that could be used for genetic diversification studies and further exploration of these strains.

Co-targeting strategy for precise, scarless gene editing with CRISPR/Cas9 and donor ssODNs in Chlamydomonas.

Programmable site-specific nucleases, such as the clustered regularly interspaced short palindromic repeat (CRISPR)/ CRISPR-associated protein 9 (Cas9) ribonucleoproteins (RNPs), have allowed creation of valuable knockout mutations and targeted gene modifications in Chlamydomonas (Chlamydomonas reinhardtii). However, in walled strains, present methods for editing genes lacking a selectable phenotype involve co-transfection of RNPs and exogenous double-stranded DNA (dsDNA) encoding a selectable marker gene. Repair of the dsDNA breaks induced by the RNPs is usually accompanied by genomic insertion of exogenous dsDNA fragments, hindering the recovery of precise, scarless mutations in target genes of interest. Here, we tested whether co-targeting two genes by electroporation of pairs of CRISPR/Cas9 RNPs and single-stranded oligodeoxynucleotides (ssODNs) would facilitate the recovery of precise edits in a gene of interest (lacking a selectable phenotype) by selection for precise editing of another gene (creating a selectable marker)—in a process completely lacking exogenous dsDNA. We used PPX1 (encoding protoporphyrinogen IX oxidase) as the generated selectable marker, conferring resistance to oxyfluorfen, and identified precise edits in the homolog of bacterial ftsY or the WD and TetratriCopeptide repeats protein 1 genes in ∼1% of the oxyfluorfen resistant colonies. Analysis of the target site sequences in edited mutants suggested that ssODNs were used as templates for DNA synthesis during homology directed repair, a process prone to replicative errors. The Chlamydomonas acetolactate synthase gene could also be efficiently edited to serve as an alternative selectable marker. This transgene-free strategy may allow creation of individual strains containing precise mutations in multiple target genes, to study complex cellular processes, pathways, or structures.

Toward best practice in cancer mutation detection with whole-genome and whole-exome sequencing.

Clinical applications of precision oncology require accurate tests that can distinguish true cancer-specific mutations from errors introduced at each step of next-generation sequencing (NGS). To date, no bulk sequencing study has addressed the effects of cross-site reproducibility, nor the biological, technical and computational factors that influence variant identification. Here we report a systematic interrogation of somatic mutations in paired tumor-normal cell lines to identify factors affecting detection reproducibility and accuracy at six different centers. Using whole-genome sequencing (WGS) and whole-exome sequencing (WES), we evaluated the reproducibility of different sample types with varying input amount and tumor purity, and multiple library construction protocols, followed by processing with nine bioinformatics pipelines. We found that read coverage and callers affected both WGS and WES reproducibility, but WES performance was influenced by insert fragment size, genomic copy content and the global imbalance score (GIV; G > T/C > A). Finally, taking into account library preparation protocol, tumor content, read coverage and bioinformatics processes concomitantly, we recommend actionable practices to improve the reproducibility and accuracy of NGS experiments for cancer mutation detection.

Simple-to-use CRISPR-SpCas9/SaCas9/AsCas12a vector series for genome editing in Saccharomyces cerevisiae.

Genome editing using the CRISPR/Cas system has been implemented for various organisms and becomes increasingly popular even in the genetically tractable budding yeast Saccharomyces cerevisiae. Because each CRISPR/Cas system recognizes only the sequences flanked by its unique protospacer adjacent motif (PAM), a certain single system often fails to target a region of interest due to the lack of PAM, thus necessitating the use of another system with a different PAM. Three CRISPR/Cas systems with distinct PAMs, namely SpCas9, SaCas9, and AsCas12a, have been successfully used in yeast genome editing. Their combined use should expand the repertoire of editable targets. However, currently available plasmids for these systems were individually developed under different design principles, thus hampering their seamless use in the practice of genome editing. Here, we report a series of Golden Gate Assembly-compatible backbone vectors designed under a unified principle to exploit the three CRISPR/Cas systems in yeast genome editing. We also created a program to assist the design of genome-editing plasmids for individual target sequences using the backbone vectors. Genome editing with these plasmids demonstrated practically sufficient efficiency in the insertion of gene fragments to essential genes (median 52.1%), the complete deletion of an open reading frame (median 78.9%), and the introduction of single amino acid substitutions (median 79.2%). The backbone vectors with the program would provide a versatile toolbox to facilitate the seamless use of SpCas9, SaCas9, and AsCas12a in various types of genome manipulation, especially those that are difficult to perform with conventional techniques in yeast genetics.

A 192\xa0bp ERV fragment insertion in the first intron of porcine TLR6 may act as an enhancer associated with the increased expressions of TLR6 and TLR1

BackgroundToll-like receptors (TLRs) play important roles in building innate immune and inducing adaptive immune responses. Associations of the TLR genes polymorphisms with disease susceptibility, which are the basis of molecular breeding for disease resistant animals, have been reported extensively. Retrotransposon insertion polymorphisms (RIPs), as a new type of molecular markers developed recently, have great potential in population genetics and quantitative trait locus mapping. In this study, bioinformatic prediction combined with PCR-based amplification was employed to screen for RIPs in porcine TLR genes. Their population distribution was examined, and for one RIP the impact on gene activity and phenotype was further evaluated.ResultsFive RIPs, located at the 3' flank of TLR3, 5' flank of TLR5, intron 1 of TLR6, intron 1 of TLR7, and 3' flank of TLR8 respectively, were identified. These RIPs were detected in different breeds with an uneven distribution among them. By using the dual luciferase activity assay a 192 bp endogenous retrovirus (ERV) in the intron 1 of TLR6 was shown to act as an enhancer increasing the activities of TLR6 putative promoter and two mini-promoters. Furthermore, real-time quantitative polymerase chain reaction (qPCR) analysis revealed significant association (p < 0.05) of the ERV insertion with increased mRNA expression of TLR6, the neighboring gene TLR1, and genes downstream in the TLR signaling pathway such as MyD88 (Myeloid differentiation factor 88), Rac1 (Rac family small GTPase 1), TIRAP (TIR domain containing adaptor protein), Tollip (Toll interacting protein) as well as the inflammatory factors IL6 (Interleukin 6), IL8 (Interleukin 8), and TNFα (Tumor necrosis factor alpha) in tissues of 30 day-old piglet. In addition, serum IL6 and TNFα concentrations were also significantly upregulated by the ERV insertion (p < 0.05).ConclusionsA total of five RIPs were identified in five different TLR loci. The 192 bp ERV insertion in the first intron of TLR6 was associated with higher expression of TLR6, TLR1, and several genes downstream in the signaling cascade. Thus, the ERV insertion may act as an enhancer affecting regulation of the TLR signaling pathways, and can be potentially applied in breeding of disease resistant animals.

Generating Nonmosaic Mutants in Xenopus Using CRISPR-Cas in Oocytes.

In CRISPR-Cas9 genome editing, double-strand DNA breaks (DSBs) primarily undergo repair through nonhomologous end joining (NHEJ), which produces insertion or deletion of random nucleotides within the targeted region (indels). As a result, frameshift mutation-mediated loss-of-function mutants are frequently produced. An alternative repair mechanism, homology-directed repair (HDR), can be used to fix DSBs at relatively low frequency. By injecting a DNA-homology repair construct with the CRISPR-Cas components, specific nucleotide sequences can be introduced within the target region by HDR. We have taken advantage of the fact that Xenopus oocytes have much higher levels of HDR than eggs to increase the effectiveness of creating precise mutations. We introduced the oocyte host transfer technique, well established for knockdown of maternal mRNA for loss-of-function experiments, to CRISPR-Cas9-mediated genome editing. The host-transfer technique is based on the ability of Xenopus oocytes to be isolated, injected with CRISPR-Cas components, and cultured in vitro for up to 5 d before fertilization. During these 5 d, CRISPR-Cas components degrade, preventing further alterations to the paternal or maternal genomes after fertilization and resulting in heterozygous, nonmosaic embryos. Treatment of oocytes with a DNA ligase IV inhibitor, which blocks the NHEJ repair pathway, before fertilization further improves the efficiency of HDR. This method allows straightforward generation of either nonmosaic F0 heterozygous indel mutant Xenopus or Xenopus with efficient, targeted insertion of small DNA fragments (73-104 nt). The germline transmission of mutations in these animals allows homozygous mutants to be obtained one generation (F1) sooner than previously reported.

Genome Editing of the Murine Calreticulin Gene Using the CRISPR/Cas9 and CPF1 System

<Background> Abnormalities of the Calreticulin (CALR) gene are frequently observed in human Myeloproliferative Neoplasms (MPN). Small deletions and insertions in the last exon of CALR, leading to frame-shift mutations, are common genetic alterations in this disease. Genome editing by the CRISPR/Cas9 (Clustered Regularly-Interspaced Short Palindromic Repeats and CRISPR-Associated proteins 9) system is one of ways to manipulate genes in normal/abnormal mammalian cells. Recently, CPF1 (CRISPR from Prevotella and Francisella 1) which is a novel single-component RNA-guided endonuclease has been identified. CPF1 cleaves double-strand DNA with a 5' overhang protruding end while Cas9 cleaves DNA with a blunt end. We have manipulated the murine calreticulin gene (calr) to introduce similar genetic defects as observed in human MPN using Cas9 and two kinds of CFP1 enzymes (AsCPF1 and LbCPF1). Efficiency of genetic alterations by the Cas9, AsCPF1 and LbCPF1 was compared. Furthermore, we used these enzymes to create Knock-In (KI) cells carrying an artificial exons in the last exon of calr .<Methods> Two guide RNAs targeting the last exon of calr were designed for Cas9, AsCPF1 and LbCPF1. Vectors encoding the Cas9 or CPF1 nuclease with GFP/YFP as a marker were transfected into murine fibroblast cell line, NIH3T3, with the vectors encoding the guide RNAs. Transfected cells were sorted on a flow cytometer using the GFP/YFP as the marker. Single cell culture was performed in 96-well plates. Six, 11, 10 clones for Cas9, AsCPF1, LbCPF1 were isolated and nucleotide sequences of the last exon of calr were confirmed by direct sequencing. Expression of the transcripts from the altered alleles were also confirmed by reverse-transcription(RT) PCR and sequencing. Furthermore, we have created a KI construct in which the last exon of calr was divided into three artificial exons and the middle exon was flanked by LoxP sequences (floxed). The KI construct was tranfected into NIH3T3 with vectors encoding the Cas9 or CFP1 as well as vectors encoding the guide RNAs. Single clones were isolated and genetic status of the last exon of calr in each clone was examined by PCR/sequencing.<Results> Nine of 11 clones for AsCPF1 (82%) and nine of 10 clones for LbCPF1 (90%) showed genetic abnormalities in the last exon of calr . Cas9 showed five manipulated clones of six clones (83%). Two, Three and Three clones were hemizygous abnormalities for Cas9, AsCPF1 and LbCPF1 respectively (33%, 27% and 30%). Three, four and four clones were compound-heterozygous abnormalities for Cas9, AsCPF1 and LbCPF1 respectively (50%, 36% and 40%). One clone for AsCPF1 and one clone for LbCPF1 were homozygous abnormalities. Majority of the genetic abnormalities were small deletion (1- 55bp). Three clones for Cas9 showed complete removal of the genome between the two target sites (168-170bp). One clone for Cas9 showed 163bp insertion in the target region. Altered messages transcribed from the manipulated alleles were confirmed by RT-PCR as well as sequencing in all the clones. As for the experiments to create the KI cells, more than 50 clones were isolated and one clone showed replacement of the last exon of calr by the KI construct. Transient expression of the Cre recommbinase by transfection efficiently deleted the floxed exon. However, we found that the KI cells expressed altered calr messages with complex sequence due to an unintentional insertion of the DNA fragment in the artificial exons.<Discussion> With the Cas9, AsCPF1 and the LbCPF1 system, we successfully introduced genetic changes of calr in murine cell line. The efficacy of introducing genomic abnormalities into calr was similar among the three endonucleases, Cas9, AsCPF1, and LbCPF1. Small deletions in calr were efficiently introduced in the cell line. However, efficiency of KI was not sufficient. Optimization may be needed to create KI cell lines using the CRISPR/Cas9 system. Nevertheless, this technique can be applied for generation of murine models carrying the pathogenic calr allele or artificial conditional exons of calr . DisclosuresNo relevant conflicts of interest to declare.

High-fidelity, efficient, and reversible labeling of endogenous proteins using CRISPR-based designer exon insertion.

Precise and efficient insertion of large DNA fragments into somatic cells using gene editing technologies to label or modify endogenous proteins remains challenging. Non-specific insertions/deletions (INDELs) resulting from the non-homologous end joining pathway make the process error-prone. Further, the insert is not readily removable. Here, we describe a method called CRISPR-mediated insertion of exon (CRISPIE) that can precisely and reversibly label endogenous proteins using CRISPR/Cas9-based editing. CRISPIE inserts a designer donor module, which consists of an exon encoding the protein sequence flanked by intron sequences, into an intronic location in the target gene. INDELs at the insertion junction will be spliced out, leaving mRNAs nearly error-free. We used CRISPIE to fluorescently label endogenous proteins in mammalian neurons in vivo with previously unachieved efficiency. We demonstrate that this method is broadly applicable, and that the insert can be readily removed later. CRISPIE permits protein sequence insertion with high fidelity, efficiency, and flexibility.

Influence of interlocking thread screws to repair simulated adult canine humeral condylar fractures.

To determine the influence of interlocking screw threads on the biomechanical properties of repaired canine humeral condylar fractures. Ex vivo biomechanical study. Thirty-six humeral condyles. Simulated fractures of the lateral aspect of the humeral condyle were stabilized by a 3.5 mm interlocking thread screw (ITS) or 3.5 mm buttress thread screw placed in lag (BTS-L) or positional fashion (BTS-P) and axially loaded at a walk, trot, 2-mm displacement, and failure cycles. Compact flute drill bits (CFBs) were used for ITS constructs and standard flute drill bits (SFB) for BTS constructs. The effects of bit type on drilling parameters and screw type on screw insertion properties and fragment stability were assessed. CFB produced a 6°C greater temperature increase (p=.042) and required 20 N higher torque (p=.003) than SFB. Insertional torque was greater for ITS than BTS-P (p=.001) and BTS-L (p=.001). Condylar fragment rotation at failure was lower in ITS (lsmean ± SE, 8.3° ± 1.9°) than BTS-L constructs (14.5° ± 2.3°, p=.011). ITS resisted greater loads (1503 ± 105 N) than BTS-P (1189 ± 99 N, p=.038) but not BTS-L (1249 ± 123 N, p=.121) constructs. Biomechanical performance of constructs was improved with ITS rather than BTS fixation. ITS can be considered for stabilization of humeral condylar fractures in adult dogs.

Dynamics of B cell repertoires and emergence of cross-reactive responses in patients with different severities of COVID-19.

SummaryIndividuals with the 2019 coronavirus disease (COVID-19) show varying severity of the disease, ranging from asymptomatic to requiring intensive care. Although monoclonal antibodies specific to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been identified, we still lack an understanding of the overall landscape of B cell receptor (BCR) repertoires in individuals with COVID-19. We use high-throughput sequencing of bulk and plasma B cells collected at multiple time points during infection to characterize signatures of the B cell response to SARS-CoV-2 in 19 individuals. Using principled statistical approaches, we associate differential features of BCRs with different disease severity. We identify 38 significantly expanded clonal lineages shared among individuals as candidates for responses specific to SARS-CoV-2. Using single-cell sequencing, we verify the reactivity of BCRs shared among individuals to SARS-CoV-2 epitopes. Moreover, we identify the natural emergence of a BCR with cross-reactivity to SARS-CoV-1 and SARS-CoV-2 in some individuals. Our results provide insights important for development of rational therapies and vaccines against COVID-19.

CRISPR/Cas9 technology in disease research and therapy: a review

Genome editing is a genetic manipulation technique that can modify DNA sequences at the genome level, including insertion, knockout, replacement and point mutation of specific DNA fragments. The ultimate principle of genome editing technology relying on engineered nucleases is to generate double-stranded DNA breaks at specific locations in genome and then repair them through non-homologous end joining or homologous recombination. With the intensive study of these nucleases, genome editing technology develops rapidly. The most used nucleases include meganucleases, zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats associated Cas proteins. Based on introducing the development and principles of above mentioned genome editing technologies, we review the research progress of CRISPR/Cas9 system in the application fields of identification of gene function, establishment of disease model, gene therapy, immunotherapy and its prospect.

MAVS splicing variants associated with TRAF3 and TRAF6 in NF-κB and IRF3 signaling pathway in large yellow croaker Larimichthys crocea

Mitochondrial antiviral signaling protein (MAVS) acts as an essential adaptor in host RIG-I-like receptors (RLRs) mediated antiviral signaling pathway. In the present study, two MAVS transcript variants, the typical form and a splicing variant, namely Lc-MAVS_tv1 and Lc-MAVS_tv2 were characterized in large yellow croaker (Larimichthys crocea). The putative Lc-MAVS_tv1 protein contains 512 aa, with an N-terminal CARD domain, a central proline-rich region, and a C-terminal transmembrane (TM) domain, whereas Lc-MAVS_tv2 contains 302 aa and lacks the C-terminal TM domain due to a premature stop in the 102 bp intron fragment insertion. Lc-MAVS_tv1 was identified as a mitochondrion localized protein whereas Lc-MAVS_tv2 exhibited an entire cytosolic distribution. Quantitative real-time PCR revealed that Lc-MAVS_tv1 mRNA was broadly expressed in examined organs/tissues and showed extremely higher level than that of Lc-MAVS_tv2, and both of them could be up-regulated under poly I:C, LPS, PGN, and Pseudomonas plecoglossicida stimulation in vivo. Interestingly, overexpression of Lc-MAVS_tv2 could induce the activation of NF-κB but not IRF3, and Lc-MAVS_tv2 co-transfected with Lc-MAVS_tv1 induced a significantly higher level of NF-κB and IRF3 promoter activity. In addition, Lc-MAVS_tv2 overexpression could enhance TRAF3 and TRAF6 mediated NF-κB activation, but suppress TRAF3 and TRAF6 mediated IRF3 activation, implying that the splicing variant Lc-MAVS_tv2 may function as an important regulator in MAVS mediated signaling pathway.

Use of Mathematical Methods for the Biosafety Assessment of Agricultural Crops.

In Russia and around the world, there are important questions regarding the potential threats to national and biological safety created by genetic technologies and the need to improve or introduce new, justified, and adequate measures for their control, regulation, and prevention. The article shows that a significant volume of the global market is occupied by five major transgenic crops, and producers are ready to switch to crops with an edited genome that has been approved in the United States, Argentina, and other countries. We propose a qualitatively new approach to the risk assessment of edited plants, “Safe Design,” and we have also developed an extremely important, fundamentally new approach to the development of methods that combine next-generation sequencing (NGS) and Bioinformatics for the assessment of the crop import biosafety. The proposed mathematical approach provides a detailed analysis of the possible insertions of DNA fragments into the genome of edited crops and a clarification of their biological significance. The developed method can be used in the rapid screening of plants for the presence of potentially dangerous genes, viral sequences, and nonspecific promoter sequences.

Pyroptosis and Redox Balance in Kidney Diseases

Significance: Kidney diseases remain a worldwide public health problem resulting in millions of deaths each year; they are characterized by progressive destruction of renal function by sustained inflammation. Pyroptosis is a lytic type of programmed cell death involved in inflammation, as well as a key fibrotic mechanism that is critical in the development of kidney pathology. Pyroptosis is induced by the cleavage of Gasdermins by various caspases and is executed by the insertion of the N-terminal fragment of cleaved Gasdermins into the plasma membrane, creating oligomeric pores and allowing the release of diverse proinflammatory products into the extracellular space. Inflammasomes are multiprotein complexes leading to the activation of caspase-1, which will cleave Gasdermin D, releasing several proinflammatory cytokines; this results in the initiation and amplification of the inflammatory response. Recent Advances: The efficacy of Gasdermin D cleavage is reduced by a change in the redox balance. Recently, several studies have shown that the attenuation of reactive oxygen species (ROS) production induced by antioxidant pathways results in a reduction of renal pyroptosis. In this review, we discuss the role of pyroptosis in the pathogenesis of chronic kidney disease (CKD) and acute kidney disease; summarize the clinical outcomes and different molecular mechanisms leading to Gasdermin activation; and examine studies about the capacity of antioxidants, particularly Nrf2 activators, to ameliorate Gasdermin activity. Future Directions: We illustrate the potential influence of the deregulation of redox balance on inflammasome activity and pyroptosis as a novel therapeutic approach for the treatment of kidney diseases. Antioxid. Redox Signal. 35, 40-60.

Comparative genomic analysis of Polypodiaceae chloroplasts reveals fine structural features and dynamic insertion sequences

BackgroundComparative chloroplast genomics could shed light on the major evolutionary events that established plastomic diversity among closely related species. The Polypodiaceae family is one of the most species-rich and underexplored groups of extant ferns. It is generally recognized that the plastomes of Polypodiaceae are highly notable in terms of their organizational stability. Hence, no research has yet been conducted on genomic structural variation in the Polypodiaceae.ResultsThe complete plastome sequences of Neolepisorus fortunei, Neolepisorus ovatus, and Phymatosorus cuspidatus were determined based on next-generation sequencing. Together with published plastomes, a comparative analysis of the fine structure of Polypodiaceae plastomes was carried out. The results indicated that the plastomes of Polypodiaceae are not as conservative as previously assumed. The size of the plastomes varies greatly in the Polypodiaceae, and the large insertion fragments present in the genome could be the main factor affecting the genome length. The plastome of Selliguea yakushimensis exhibits prominent features including not only a large-scale IR expansion exceeding several kb but also a unique inversion. Furthermore, gene contents, SSRs, dispersed repeats, and mutational hotspot regions were identified in the plastomes of the Polypodiaceae. Although dispersed repeats are not abundant in the plastomes of Polypodiaceae, we found that the large insertions that occur in different species are mobile and are always adjacent to repeated hotspot regions.ConclusionsOur results reveal that the plastomes of Polypodiaceae are dynamic molecules, rather than constituting static genomes as previously thought. The dispersed repeats flanking insertion sequences contribute to the repair mechanism induced by double-strand breaks and are probably a major driver of structural evolution in the plastomes of Polypodiaceae.

Development of a Novel Orthotopic Gastric Cancer Mouse Model

BackgroundGastric cancer metastasis is a highly fatal disease with a five-year survival rate of less than 5%. One major obstacle in studying gastric cancer metastasis is the lack of faithful models available. The cancer xenograft mouse models are widely used to elucidate the mechanisms of cancer development and progression. Current procedures for creating cancer xenografts include both heterotopic (i.e., subcutaneous) and orthotopic transplantation methods. Compared to the heterotopic model, the orthotopic model has been shown to be the more clinically relevant design as it enables the development of cancer metastasis. Although there are several methods in use to develop the orthotopic gastric cancer model, there is not a model which uses various types of tumor materials, such as soft tissues, semi-liquid tissues, or culture derivatives, due to the technical challenges. Thus, developing the applicable orthotopic model which can utilize various tumor materials is essential.ResultsTo overcome the known limitations of the current orthotopic gastric cancer models, such as exposure of tumor fragments to the neighboring organs or only using firm tissues for the orthotopic implantation, we have developed a new method allowing for the complete insertion of soft tissue fragments or homogeneously minced tissues into the stomach submucosa layer of the immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mouse. With this completely-closed transplantation method, tumors with various types of tissue may be used to establish orthotopic gastric cancer models without the risks of exposure to nearby organs or cell leakage. This surgical procedure was highly reproducible in generating forty-eight mouse models with a surgery success rate of 96% and tumor formation of 93%. Among four orthotopic patient-derived xenograft (PDX) models that we generated in this study, we verified that the occurrence of organotropic metastasis in either the liver or peritoneal cavity was the same as that of the donor patients.ConclusionHere we describe a new protocol, step by step, for the establishment of orthotopic xenograft of gastric cancer. This novel technique will be able to increase the use of orthotopic models in broader applications for not only gastric cancer research but also any research related to the stomach microenvironment.

Genome editing technologies: CRISPR, LEAPER, RESTORE, ARCUT, SATI, and RESCUE.

Genome editing technologies include techniques used for desired genetic modifications and allow the insertion, modification or deletion of specific DNA fragments. Recent advances in genome biology offer unprecedented promise for interdisciplinary collaboration and applications in gene editing. New genome editing technologies enable specific and efficient genome modifications. The sources that inspire these modifications and already exist in the genome are DNA degradation enzymes and DNA repair pathways. Six of these recent technologies are the clustered regularly interspaced short palindromic repeats (CRISPR), leveraging endogenous ADAR for programmable editing of RNA (LEAPER), recruiting endogenous ADAR to specific transcripts for oligonucleotide-mediated RNA editing (RESTORE), chemistry-based artificial restriction DNA cutter (ARCUT), single homology arm donor mediated intron-targeting integration (SATI), RNA editing for specific C-to-U exchange (RESCUE). These technologies are widely used from various biomedical researches to clinics, agriculture, and allow you to rearrange genomic sequences, create cell lines and animal models to solve human diseases. This review emphasizes the characteristics, superiority, limitations, also whether each technology can be used in different biological systems and the potential application of these systems in the treatment of several human diseases.

Development of SSR Markers Associated with Polima CMS Fertility Restorer in &lt;i&gt;Brassica napus&lt;/i&gt; L. and Its Application

The Polima system of cytoplasmic male sterility (Pol CMS) was widely used for hybrid seed production, but with high probability of occurrence of maternal false hybrid due to the fertility of Polima CMS (Pol CMS) line greatly affected by the ambient temperature, therefore, the detection of maternal false hybrids is the key to purity identification. In present study, the SSR markers were designed based on the flanking sequences of Pol CMS restorer gene&nbsp; Rfp , and application of SSR markers in hybrid seed purity test of rapeseed. The main results were as follows: the sequences of &nbsp;Rfp&nbsp; were subjected to basic local alignment search tool queries against the&nbsp; Brassica rapa &nbsp;genome to determine chromosome positions, and 32 SSR markers were developed based on the upstream and downstream sequences of&nbsp; Rfp . Among the 32 SSR markers, one codominant marker was polymorphic between Pol CMS sterile and restorer line, the PCR product length was 162 bp in Pol CMS sterile lines, and 184 bp in restorer lines. Based on the PCR sequence, two insert fragments (15 bp and 7 bp) were identified in Pol CMS restorer lines. The SSR marker co-segregated with plant fertility of individuals in the &ldquo;fengyou737&rdquo; F 2 &nbsp;population. The purity of &ldquo;fengyou737&rdquo;, &ldquo;fengyou320&rdquo; and &ldquo;fengyou958&rdquo; were identified by means of PCR-SSR markers and field-planting, and the results of two ways were highly consistent, indicated that the codominant SSR marker tightly linked to&nbsp; Rfp , and the marker can detect paternal and maternal false hybrids. The present findings will provide technical assistance for hybrid seed purity test and genetic diversity analysis in rapeseed.

Cloning and over Expression Studies of Ovine Somatotropin cDNA of Kajli (sheep breed) in a Prokaryotic System.

Genetic studies including the quest, cloning and expression of genes encoding proteins responsible for various vital physiological processes and beneficial characteristics of economic perspective have made the biotechnology research progressively auspicious. Due to its great zootechnical and industrial importance somatotropin gene have been cloned from various animal species. Current study was designed to clone mature ovine growth hormone complementary DNA (oGH cDNA) of a sheep breed, Kajli and carry out over expression studies of cloned GH cDNA in a suitable prokaryotic expression system. Sheep GH cDNA was cloned in T/A (thymine / adenine) vector with signal peptide and confirmed by nested polymerase chain reaction (PCR) and restriction digestion. The gene was then ligated in pLEX expression vector and restricted plasmids showed a fragment insert of ~ 600 bps. Restriction analysis confirmed positive clones, were induced for protein expression analysis. The pET vectors (plasmid for expression by T7 RNA polymerase) have an isopropylthio-β-galactoside (IPTG) inducible strong T7 promoter and Escherichia coli expression strain of BL21 (DE3) pLysS contains DNA fragment from T7 phage which harbors RNA polymerase. Therefore, for expressing recombinant proteins, cells were induced with various IPTG concentrations to optimize expression levels. Cells were induced with different IPTG concentrations (0.1 to 0.8 mM) followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Results indicated maximum expression level of oGH at 5 hrs after induction of cells with 0.3 mM IPTG concentration with a molecular weight of 22 kDa. As for as cellular localization of protein is concerned accumulation of expressed oGH is observed in inclusion bodies. The successful expression of the cloned GH cDNA of sheep confirmed the functional viability of the clone. The above mentioned technique of genetic engineering has provided to boost the dairy industry by the production of large quantities of recombinant bovine somatotropin (rbST).