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Moppett, John, Jerry Hancock, Marc Duez, Jack Bartram, Gary Wright, Mike Hubank, Paul Archer, Helen Williamson, and Stephanie Wakeman. "Artificial DNA Templates for the Correction of PCR Bias in Next Generation Sequencing Based MRD Analysis for Childhood Acute Lymphoblastic Leukaemia: The Influence of Secondary DNA Structure." Blood 128, no. 22 (December 2, 2016): 4078. http://dx.doi.org/10.1182/blood.v128.22.4078.4078.
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Abstract PCR bias is a potential confounder for PCR-based NGS-MRD quantitation. A method was published using artificial DNA constructs (gBlocks, IDT Technologies, Coralville, IO) to assess PCR primer bias and correct for it(1). We sought to confirm those findings by assessing PCR bias of the Biomed-2 TRG primer set. 36 synthetic DNA templates of 495bp length gBlocks combining each TRG V (V 2,3,4,5,7,8,9,10,11) & J gene (JG1-01, JG1-02, JP1, JP2) amplified by the Biomed-2 primer set were synthesised containing external MiSeq flow cell adaptor sequence (universal primer (UP)) binding sites, VG and JG sequence and junctional regions. Barcodes inserted internal to the universal primer sites and centrally enabled accurate template identification (fig 1). Individual gBlocks supplied at a nominal concentration of 10ng/uL (30nM) were quantitated by TapeStation (Agilent, Santa Clara, CA) and KAPA (Kapa Biosystems, Wilmington, MA) methodologies and pooled (unadjusted). gBlockswithin the pool were further quantitated by both 6 and 8 cycles of PCR with universal MiSeq adaptor primers. Next the gBlock pool was amplified in triplicate in a 2-stage PCR process: 1) Using Biomed-2 TRG primers that contained partial MiSeq adaptor sequences. The resulting products were purified with Agencourt AMPure XP beads (Beckman Coulter, Jersey City, NJ). 2) These amplicons were further amplified by primers containing indices and full MiSeq adaptor sequences. After purification, amplicons were Qubit (InvitroGen, Carlsbad, CA) and TapeStation analysed for normalisation and to construct the sequencing library, which was KAPA quantitated to ensure ideal cluster density. Bi-directional sequencing was performed using an Illumina MiSeq 500 cycle cartridge and Nano Reagent Kit (Illumina, San Diego, CA). Bioinformatics analysis was performed using the Vidjil platform (Bonsai team, CRIStAL, Lille, France). TapeStation quantitation showed a mean concentration of 3.3ng/uL (10nM) (range 1.1-6.3ug/uL). KAPA quantitation by contrast showed low quantities of all gBlocks (2nM, 0.67ug/uL) with the exception of all VG9 and most VG10 constructs (16nM, 5ug/uL)(fig 2a). Comparison of 6 vs 8 cycles of amplification using universal primers showed good correlation (R2=0.991) confirming that PCR cycle length does not affect the UP amplification (fig 2b), but there was an 8.5-fold variation in gBlock quantitation by UP PCR. There was no correlation between NGS quantitation and with either TapeStation or KAPA quantitation. Family specific V & J gene amplification efficiency for the Biomed-2 primers is shown in fig 3. Amplification efficiencies of V gene primers were <2-fold different with the exception of VY9 and VY10 which were 5-fold more efficient. Amplification efficiency for J gene primers showed <2-fold differences. This study has shown that there are clear PCR efficiency differences between the V primers in the Biomed-2 primer set, with VG9 and 10 being 5x more efficient. This could be adjusted for by reducing the concentrations of the VG9/10 primers. However, the variable results of the 3 methods used to quantitate the artificial templates raises significant questions about the rationale. TapeStation quantitation is not PCR based and produced broadly equivalent (but lower than anticipated) gBlock concentrations for the individual constructs. PCR based KAPA showed very low concentrations in all but the VG9/VG10 templates. It is notable that this exactly parallels the results for the locus specific PCR. The most plausible explanation is that unexpected secondary structure reduces the amplifiability of the templates in PCR reactions. The near 10-fold variation in template concentration as assessed by UP NGS-PCR is concerning, suggesting significant secondary structure effects, though not dissimilar to the 5-fold difference seen in the published method(1). It is impossible to know if the postulated secondary structure effect that we see is replicated in vivo. In that regard it is reassuring that published results of NGS-MRD appear to correlate well with RQ-PCR results. In conclusion, secondary structure issues potentially affect the in vitro method for eliminating PCR bias. Within the EuroClonality consortium, studies at other loci are underway to confirm these preliminary findings, and to design primer sets with minimal risk of PCR bias. 1. Carlson CS, Emerson RO, Sherwood AM et al. Nat Commun. 2013;4:2680. Disclosures Moppett: Jazz Pharmaceuticals: Honoraria.
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Sharma, Ashish Ranjan, Chiranjib Chakraborty, Sang-Soo Lee, Garima Sharma, Jeong Kyo Yoon, C. George Priya Doss, Dong-Keun Song, and Ju-Suk Nam. "Computational Biophysical, Biochemical, and Evolutionary Signature of Human R-Spondin Family Proteins, the Member of Canonical Wnt/β-Catenin Signaling Pathway." BioMed Research International 2014 (2014): 1–22. http://dx.doi.org/10.1155/2014/974316.
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In human, Wnt/β-catenin signaling pathway plays a significant role in cell growth, cell development, and disease pathogenesis. Four human (Rspo)s are known to activate canonical Wnt/β-catenin signaling pathway. Presently, (Rspo)s serve as therapeutic target for several human diseases. Henceforth, basic understanding about the molecular properties of (Rspo)s is essential. We approached this issue by interpreting the biochemical and biophysical properties along with molecular evolution of (Rspo)s thorough computational algorithm methods. Our analysis shows that signal peptide length is roughly similar in (Rspo)s family along with similarity in aa distribution pattern. In Rspo3, four N-glycosylation sites were noted. All members are hydrophilic in nature and showed alike GRAVY values, approximately. Conversely, Rspo3 contains the maximum positively charged residues while Rspo4 includes the lowest. Four highly aligned blocks were recorded through Gblocks. Phylogenetic analysis shows Rspo4 is being rooted with Rspo2 and similarly Rspo3 and Rspo1 have the common point of origin. Through phylogenomics study, we developed a phylogenetic tree of sixty proteins (n=60) with the orthologs and paralogs seed sequences. Protein-protein network was also illustrated. Results demonstrated in our study may help the future researchers to unfold significant physiological and therapeutic properties of (Rspo)s in various disease models.
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Billones-Baaijens, Regina, José Ramón Úrbez-Torres, Meifang Liu, Matthew Ayres, Mark Sosnowski, and Sandra Savocchia. "Molecular Methods to Detect and Quantify Botryosphaeriaceae Inocula Associated With Grapevine Dieback in Australia." Plant Disease 102, no. 8 (August 2018): 1489–99. http://dx.doi.org/10.1094/pdis-11-17-1854-re.
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Botryosphaeria dieback, caused by species of Botryosphaeriaceae, is an important grapevine trunk disease in Australia. Inocula produced by the pathogens are primarily dispersed by rain splash and wind and infect pruning wounds leading to cankers, dieback, and eventually death of vines. The objective of this study was to develop molecular tools to detect and quantify Botryosphaeriaceae inocula from the environment. These tools are essential for investigating spore dispersal patterns of Botryosphaeriaceae pathogens in Australian vineyards. DNA extraction protocols were evaluated and one modified protocol was found suitable for extracting Botryosphaeriaceae DNA from artificially and naturally inoculated Burkard volumetric spore sampler tapes. Multispecies primers and a hydrolysis probe for quantitative PCR (qPCR) were further developed to detect and quantify Botryosphaeriaceae inocula from environmental samples. Specificity tests showed that the multispecies primers were able to amplify the DNA of 10 Botryosphaeriaceae species (58 isolates) found in Australia while none of the 27 nontarget fungal species (90 isolates) tested were amplified. The qPCR assay was suitable for amplifying purified DNA, synthetic DNA fragments (gBlocks), and mixed DNA from spore trap tapes. The qPCR method developed in this study was shown to be rapid and sensitive in detecting Botryosphaeriaceae inocula from the environment using spore traps.
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Lastra González, David, Vojtech Baláž, Jiří Vojar, and Petr Chajma. "Dual Detection of the Chytrid Fungi Batrachochytrium spp. with an Enhanced Environmental DNA Approach." Journal of Fungi 7, no. 4 (March 30, 2021): 258. http://dx.doi.org/10.3390/jof7040258.
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Environmental DNA (eDNA) is becoming an indispensable tool in biodiversity monitoring, including the monitoring of invasive species and pathogens. Aquatic chytrid fungi Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal) are major threats to amphibians. However, the use of eDNA for detecting these pathogens has not yet become widespread, due to technological and economic obstacles. Using the enhanced eDNA approach (a simple and cheap sampling protocol) and the universally accepted qPCR assay, we confirmed the presence of Bsal and Bd in previously identified sites in Spain, including four sites that were new for Bsal. The new approach was successfully tested in laboratory conditions using manufactured gene fragments (gBlocks) of the targeted DNA sequence. A comparison of storage methods showed that samples kept in ethanol had the best DNA yield. Our results showed that the number of DNA copies in the Internal Transcribed Spacer region was 120 copies per Bsal cell. Eradication of emerging diseases requires quick and cost-effective solutions. We therefore performed cost-efficiency analyses of standard animal swabbing, a previous eDNA approach, and our own approach. The procedure presented here was evaluated as the most cost-efficient. Our findings will help to disseminate information about efforts to prevent the spread of chytrid fungi.
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Yulita, Dina Sri, Aqwin Polosoro, Atmitri Sisharmini, Aniversari Apriana, Febi Nurilmala, and Kurniawan Rudi Trijatmiko. "Construction of Binary Vector and Transformation of Synthetic LcCsp Gene into Nipponbare Rice Genome by Agrobacterium tumefaciens Transformation Method." Jurnal AgroBiogen 16, no. 1 (July 17, 2020): 25. http://dx.doi.org/10.21082/jbio.v16n1.2020.p25-34.
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<p>Cold shock protein (Csp) is an essential bacterial protein for increasing abiotic stress tolerance, especially cold stress. Several studies discovered that overexpression of the gene successfully improves the tolerances of several types of plant not only under cold stress, but also other abiotic stresses, e.g. hot and drought conditions. The objectives of this study were to construct a binary vector containing the LcCsp gene modified from Lactobacillus casei and transform the gene into Nipponbare rice genome. The native LcCsp gene sequence, however, has low GC content (46.7%) while rice as transformation target plant has 52% GC content. The native LcCsp gene sequence, therefore, was optimized to the level close to 52.7% similar to GC content of the rice genome. This LcCsp gene was synthesized by using DNA printing technology (gBlocks® Gene Fragments Entry, IDT). The synthetic LcCsp gene was successfully inserted into the pCAMBIA1300-int binary vector driven by Ubiquitin1 promoter and NOS terminator. The T-DNA cassette was successfully transformed into Nipponbare rice genome by Agrobacterium tumefaciens strain LBA4404 using immature embryo transformation protocol. A total of 51 T0 Nipponbare lines survived from hygromycin selections and 21 lines were successfully acclimatized. Molecular analysis of the candidate lines showed that all Nipponbare transgenic putative lines contain the LcCsp gene demonstrating high transformation efficiency of 11.8%. The rice lines resulted from this study should be further analyzed and might be useful for developing rice transgenic lines tolerance to heat, drought, or saline stress condition.</p>
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Dittmann, Isabel L., Daniel Cuadrado, Maria Teresa Aguado, Carolina Noreña, and Bernhard Egger. "Polyclad phylogeny persists to be problematic." Organisms Diversity & Evolution 19, no. 4 (September 16, 2019): 585–608. http://dx.doi.org/10.1007/s13127-019-00415-1.
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Abstract Two conflicting morphological approaches to polyclad systematics highlight the relevance of molecular data for resolving the interrelationships of Polycladida. In the present study, phylogenetic trees were reconstructed based on a short alignment of the 28S rDNA marker gene with 118 polyclad terminals (24 new) including 100 different polyclad species from 44 genera and 22 families, as well as on a combined dataset using 18S and 28S rDNA genes with 27 polyclad terminals (19 new) covering 26 different polyclad species. In both approaches, Theamatidae and Cestoplanidae were included, two families that have previously been shown to switch from Acotylea to Cotylea. Three different alignment methods were used, both with and without alignment curation by Gblocks, and all alignments were subjected to Bayesian inference and maximum likelihood tree calculations. Over all trees of the combined dataset, an extended majority-rule consensus tree had weak support for Theamatidae and Cestoplanidae as acotyleans, and also the cotylean genera Boninia, Chromyella and Pericelis appeared as acotyleans. With the most inclusive short 28S dataset, on the other hand, there is good support for the aforementioned taxa as cotyleans. Especially with the short 28S matrix, taxon sampling, outgroup selection, alignment method and curation, as well as model choice were all decisive for tree topology. Well-supported parts of the phylogeny over all trees include Pseudocerotoidea, Prosthiostomoidea, Stylochoidea, Leptoplanoidea and Cryptoceloidea, the latter three with new definitions. Unstable positions in the tree were found not only for Theamatidae, Cestoplanidae, Boninia, Chromyella and Pericelis, but also for Anonymus, Chromoplana and Cycloporus.
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Van der Straeten, Jona, Wouter De Brouwer, Emmanuelle Kabongo, Marie-Françoise Dresse, Karel Fostier, Rik Schots, Ivan Van Riet, and Marleen Bakkus. "Validation of a PCR-Based Next-Generation Sequencing Approach for the Detection and Quantification of Minimal Residual Disease in Acute Lymphoblastic Leukemia and Multiple Myeloma Using gBlocks as Calibrators." Journal of Molecular Diagnostics 23, no. 5 (May 2021): 599–611. http://dx.doi.org/10.1016/j.jmoldx.2021.01.009.
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Giribet, Gonzalo, and Gregory D. Edgecombe. "Stable phylogenetic patterns in scutigeromorph centipedes (Myriapoda : Chilopoda : Scutigeromorpha): dating the diversification of an ancient lineage of terrestrial arthropods." Invertebrate Systematics 27, no. 5 (2013): 485. http://dx.doi.org/10.1071/is13019.
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Although stable and well-supported relationships are in place for the three main clades (families) of Scutigeromorpha, the interrelationships of particular taxa within the most diverse family, Scutigeridae, are less clearly resolved. Novel molecular data for taxa from Mesoamerica, the Caribbean, southern Africa, New Guinea and previously unsampled parts of the Pacific are incorporated into phylogenetic analyses. Relationships across the tree are stable under variable analytical conditions, whether these are homology-based (multiple sequence alignment versus implied alignment; untrimmed versus trimmed datasets) or method-based (parsimony versus maximum likelihood). Hypervariable regions, contrary to common belief, add phylogenetic structure to the data, as measured by the increased support for many nodes when compared with the same alignments trimmed with Gblocks. Our analyses show that a Yule-3-rate model best explained the diversification of Scutigeromorpha during their 400 million years of history. More complete molecular data for the New Guinea genus Ballonema stabilise its position as sister group to Thereuoneminae. To reconcile scutigeromorph systematics with the phylogeny, the monotypic genus Madagassophora Verhoeff, 1936, is placed in synonymy with Scutigerina Silvestri, 1901 (n. syn.), its type species M. hova becoming Scutigerina hova (de Saussure & Zehntner, 1902) new comb. (from Scutigera), and Lassophora Verhoeff, 1905, is re-established for an Afro-Malagasy clade containing Lassophora nossibei (de Saussure & Zehntner, 1902) new comb. (from Scutigera) and a newly sequenced species from Mozambique that diverged at the base of the lineage to Thereuoneminae. The dated phylogeny of Scutigeromorpha is more consistent with ancient vicariant splits between Madagascar–southern Africa and Australia–New Caledonia than with younger dispersal scenarios, though some geologically young Pacific islands that harbour lineages dating to the Cretaceous demonstrate the potential for trans-oceanic dispersal.
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Spate, A. M., K. M. Whitworth, C. W. O'Gorman, A. K. Byrne, R. S. Prather, and K. D. Wells. "26 THE USE OF PAIRED CRISPR GUIDE RNAs AND THE Cas9 SYSTEM DOES NOT ALWAYS PRODUCE SITE SPECIFIC DELETIONS OF GENE SEQUENCE IN PORCINE CELL AND EMBRYO CULTURE." Reproduction, Fertility and Development 29, no. 1 (2017): 120. http://dx.doi.org/10.1071/rdv29n1ab26.
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The use of the CRISPR/Cas9 system has become increasingly popular for creating gene edits in both cell and embryo culture. High specificity and efficiency of editing as well as low cost and ease of use has helped to promote its use. We hypothesised that by using multiple CRISPR guides at one time, we could quickly create exact deletions spanning greater areas of sequence. A total of 5 candidate genes (A, B, C, D, E) were targeted for deletions ranging in size of 74 to 551 bp. All modifications were created through the co-injection of 2 CRISPR guide RNAs with Cas9 RNA into in vitro-produced presumptive porcine zygotes. The CRISPR guides were created using gBlocks containing the T7 promoter sequence, 18–24 bp of CRISPR guide RNA, and 85 bp of tracer RNA. The RNA structure of each guide was reviewed using RNA Folding Form as well as offsite cutting using NCBI Blast. CRISPR guide RNA pairs (20 ng μL−1) and Cas9 RNA (20 ng μL−1) were co-injected (1–3 ρl) into the cytoplasm of IVF produced porcine zygotes using the FemtoJet 4i injector. Following injections, the zygotes were cultured in vitro for 5–6 days, and viable blastocyst or morula were selected for embryo transfer into recipient gilts. Resulting pigs were assayed for expected modifications using PCR. Pigs were considered modified if an insertion or deletion was measured by gel electrophoresis and DNA sequencing. Only one pair of CRISPR guides was injected per zygote, resulting in an individual PCR assay for the gene of interest. In total, 42 live piglets were born, 24 of which were edited, yielding 57% modification. When expected modifications v. observed were analysed, only 4 of 24 pigs (16%) produced the predicted modification on at least one allele. Of the remaining 20 pigs, several showed more than one form of modification. Insertions of ranging from 1 to 400 bp were detected in 10 pigs, 9 pigs formed biallelic modifications, 6 pigs produced altered sequence for greater than 2 alleles (mosaic), 6 pigs had deletions larger than the expected ranging from 11 to 1739 bp, and 14 had deletions smaller than the expected. Due to the absence of plasmid during injections, the insertions observed contained repetitive elements from the gene being modified as well as random additional bases. Additionally, CRISPR pairs were used in cell culture of porcine fibroblast modifying gene F, where they produced 6 different deletions ranging from the expected 63b to 617 bp. We recognise that the cutting efficiency of each CRISPR guide was not measured, as our goal was to create the expected deletions from pairs of CRISPR guides. We acknowledge our hypothesis was incorrect, as this data indicates that the CRISPR/Cas9 system is a very useful tool for gene editing, however it can induce unexpected modifications when used in pairs, in cell and embryo culture. Study was supported by funding from Food for the 21st Century and NIH (U42OD011140).
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Vannuffel, Pascal, Luana Bavaro, Friedel Nollet, Asena Aynaci, Margherita Martelli, Helena Devos, Céline De Rop, and Simona Soverini. "Droplet Digital PCR Phasing (DROP-PHASE): A Novel Method for Straightforward Detection of BCR-ABL1 Compound Mutations in Tyrosine Kinase Inhibitors Resistant Chronic Myeloid Leukemia (CML) and Acute Lymphoblastic Leukemia (ALL)." Blood 134, Supplement_1 (November 13, 2019): 4660. http://dx.doi.org/10.1182/blood-2019-123409.
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Chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL) are, respectively, a myeloproliferative and a lymphoproliferative neoplasm that can be characterized by the chimeric fusion oncogene BCR-ABL1. Tyrosine Kinase Inhibitors (TKI) are the standard therapy for patients with CML/ALL. However, mutations of the BCR-ABL1 kinase domain constitute a major cause of treatment failure in CML and ALL receiving TKI therapy. While 2nd and 3rd generation TKI have proven their efficacy against mutated BCR-ABL1-mediated clonal expansion, the presence of compound mutations can produce high level of resistance to these TKIs. Even the last addition to the TKI armamentarium, ponatinib, remains ineffective against some BCR-ABL1 compound mutations (Zabriskie, M.S., et al., BCR-ABL1 Compound Mutations Combining Key Kinase Domain Positions Confer Clinical Resistance to Ponatinib in Ph Chromosome-Positive Leukemia. Cancer Cell, 2014. 26(3):p.428-442). Therefore, the distinction between compound (different mutations present on 1 unique malignant clone) and polyclonal mutations (different mutations present on 2 or more different clones) is of great clinical importance in order to select the most suitable treatment and to estimate outcomes. The objective of this study is to determine in a straightforward way whether BCR-ABL1 mutations discovered by Next Generation Sequencing are compound mutations or polyclonal mutations. A simple proof-of-concept experiment was first performed by using 3 synthetic oligonucleotides (gBlocks, IDT) mimicking the presence of compound mutations versus polyclonal mutations in resistant leukemia cells. The first oligo harbored the M237I mutation, the second oligo mutations E255K, E279K, V299L, T315I, F359V, A380S, H396R, S417Y, F459K and F486S and the third one contained all the mutations. Dual-color probes assays have been set up to target specifically 2 different mutations. Mixtures of 2 oligonucleotides harboring 1 mutation each versus 1 oligonucleotide harboring 2 mutations have been compared by performing duplex droplet digital PCR (ddPCR) reactions on the Bio-Rad ddPCR QX200 System. Linkage detection is based on the observation that the presence of 2 targets on the same DNA molecule increases the number of double-positive droplets relative to the number expected due to chance. Automatic linkage evaluation was made by the QuantaSoft Software and mathematical calculations refer to (Regan, J.F., et al., A rapid molecular approach for chromosomal phasing. PLoS One, 2015. 10(3): p. e0118270). The first experiment successfully validated the detection of mutations residing on two different oligonucleotides (polyclonal mutations) versus mutations on the same molecule (compound mutations). When performing serial dilutions of 2 oligonucleotides containing different mutations, a sensitivity of 10%:90% was achieved with a good linearity (r2=0.97). Mixing experiment also showed that ddPCR phasing could distinguish between a mixture of compound and polyclonal mutations versus and the sole presence of polyclonal mutations at the same sensitivity and linearity levels. Moreover, no influence of the genomic distance between mutations (from position 255 to position 562) was observed. The strategy was further applied to 20 clinical samples from CML/ALL patients characterized by multiple resistance mutations. Drop-phase is a rapid (< 4 hours), scalable (100 samples), technically easy to perform and cost-effective method. This strategy will help to identify compound mutations in patients with TKI-resistant CML/ALL and allow to modulate the patient's drug strategy and to prevent progression and therapeutic failure. Disclosures Vannuffel: Incyte: Consultancy. Soverini:Incyte: Consultancy.
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Whitworth, K. M., S. L. Murphy, J. A. Benne, L. D. Spate, E. Walters, R. Hickey, S. L. Nyberg, K. D. Wells, and R. S. Prather. "25 GENOME EDITING OF SOMATIC CELL NUCLEAR TRANSFER DERIVED ZYGOTES BY CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR)/Cas9 GUIDE RNA INJECTION." Reproduction, Fertility and Development 28, no. 2 (2016): 142. http://dx.doi.org/10.1071/rdv28n2ab25.
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Recent applications of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system have greatly improved the efficiency of genome editing in pigs. However, in some cases, genetically modified pig models need an additional modification to improve their application. The objective of this experiment was to determine whether a combination of somatic cell NT (SCNT) by using a previously modified donor cell line and subsequent zygote injection with CRISPR/Cas9 guide RNA to target a second gene would result in embryos and offspring successfully containing both modifications. Fibroblast cell lines were collected from fumarylacetoacetate hydrolase deficient (FAH–/–) fetuses and used as the donor cell line. Somatic cell NT was performed by standard technique. A CRISPR guide RNA specific for recombination activating gene 2 (RAG2) was designed and in vitro transcribed from a synthesised gBlock (IDT) containing a T7 promoter sequence, the CRISPR Guide RNA (20 bp), and 85 bp of tracer RNA. The gBlock was PCR amplified with Q5 polymerase (NEB, Ipswich, MA, USA) and in vitro transcribed with the MEGAshortscript™ T7 Transcription Kit (Life Technologies, Grand Island, NY, USA). Guide RNA (20 ng μL–1) and polyadenylated Cas9 (20 ng μL–1, Sigma, St. Louis, MO, USA) were co-injected into the cytoplasm of SCNT zygotes at 14 to 16 h after fusion and activation. Injected SCNT were then cultured in vitro in PZM3 + 1.69 mM arginine medium (MU1) to Day 5. Three embryo transfers were performed surgically into recipient gilts on Day 4 or 5 of oestrus (50, 62, or 70 embryos per pig) to evaluate in vivo development. The remaining embryos were cultured in MU1 to Day 7 and analysed for the presence of modifications to the RAG2 gene. Embryos were classified as modified if they contained an INDEL as measured by both gel electrophoresis and DNA sequencing of PCR amplicons spanning the targeted exon. The RAG2 modification rate was 83.3% (n = 6), of which 50% (n = 3) of the embryos contained biallelic modifications. All control embryos contained a wild-type RAG2 gene (n = 5). Embryo transfer resulted in a 33.3% pregnancy rate (1/3). The combination of SCNT and CRISPR/Cas9 zygote injection can be a highly efficient tool to successfully create pig embryos with an additional modification. This additional technique further improves the usefulness of already created genetically modified pig models. This study was funded by the National Institutes of Health via U42 OD011140.
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García-Venzor, Alfredo, Edna Ayerim Mandujano-Tinoco, Araceli Ruiz-Silvestre, José Manuel Sánchez, Floria Lizarraga, Cecilia Zampedri, Jorge Melendez-Zajgla, and Vilma Maldonado. "lncMat2B regulated by severe hypoxia induces cisplatin resistance by increasing DNA damage repair and tumor-initiating population in breast cancer cells." Carcinogenesis 41, no. 11 (July 25, 2020): 1485–97. http://dx.doi.org/10.1093/carcin/bgaa078.
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Abstract Multicellular tumor spheroids (MCTSs) constitute a three-dimensional culture system that recapitulates the in vivo tumor microenvironment. Tumor cells cultured as MCTSs present antineoplastic resistance due to the effect of microenvironmental signals acting upon them. In this work, we evaluated the biological function of a new microenvironment-regulated long non-coding RNA, lncMat2B, in breast cancer. In MCTSs, the expression of lncMat2B presented an increase and a zonal heterogeneity, as it was expressed principally in quiescent cells of hypoxic regions of the MCTSs. As expected, functional assays supported the role of severe hypoxia in the regulation of lncMat2B. Moreover, gain- and loss-of-function assays using a transcriptional silencing CRISPR/Cas9 system and gBlock revealed that lncMAT2B regulates the tumor-initiating phenotype. Interestingly, lncMat2B is overexpressed in a cisplatin-resistant MCF-7 cell line, and its ectopic expression in wild type MCF-7 cells increased survival to cisplatin exposure by reducing DNA damage and reactive oxygen species accumulation. lncMAT2B is a possible link between severe hypoxia, tumor-initiating phenotype and drug resistance in breast cancer cells.
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Warang, Anushri, Michael Zhang, Shuping Zhang, and Zhenyu Shen. "A panel of real-time PCR assays for the detection of Bourbon virus, Heartland virus, West Nile virus, and Trypanosoma cruzi in major disease–transmitting vectors." Journal of Veterinary Diagnostic Investigation, August 20, 2021, 104063872110395. http://dx.doi.org/10.1177/10406387211039549.
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Vector-borne pathogens, such as Bourbon virus (BRBV), Heartland virus (HRTV), West Nile virus (WNV), and Trypanosoma cruzi (TCZ) are a great threat to public health and animal health. We developed a panel of TaqMan real-time PCR assays for pathogen surveillance. PCR targets were selected based on nucleic acid sequences deposited in GenBank. Primers and probes were either designed de novo or selected from publications. The coverages and specificities of the primers and probes were extensively evaluated by performing BLAST searches. Synthetic DNA or RNA fragments (gBlocks) were used as PCR templates in initial assay development and PCR positive controls in subsequent assay validation. For operational efficiency, the same thermocycling profile was used in BRBV, HRTV, and WNV reverse-transcription quantitative PCR (RT-qPCR) assays, and a similar thermocycling profile without the initial reverse-transcription step was used in TCZ qPCR. The assays were optimized by titrating primer and probe concentrations. The analytical sensitivities were 100, 100, 10, and 10 copies of gBlock per reaction for BRBV (Cq = 36.0 ± 0.7), HRTV (Cq = 36.6 ± 0.9), WNV (Cq = 35.5 ± 0.4), and TCZ (Cq = 38.8 ± 0.3), respectively. PCR sensitivities for vector genomic DNA or RNA spiked with gBlock reached 100, 100, 10, and 10 copies per reaction for BRBV, HRTV, WNV, and TCZ, respectively. PCR specificity evaluated against a panel of non-target pathogens showed no significant cross-reactivity. Our BRBV, HRTV, WNV, and TCZ PCR panel could support epidemiologic studies and pathogen surveillance.
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Bidla, Gawa, David S. Rosenblatt, and Brian M. Gilfix. "Identification of Variants in Alpha-1-Antitrypsin by High Resolution Melting." Journal of Applied Laboratory Medicine, December 17, 2020. http://dx.doi.org/10.1093/jalm/jfaa191.
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Abstract Background Alpha-1-antitrypsin deficiency (AATD) is one of the most common hereditary disorders occurring in populations of European origin and is due to variants in SERPINA1, which encodes a protease inhibitor of neutrophil elastase, limiting lung damage from this enzyme. The World Health Organization has recommended that individuals with chronic obstructive pulmonary disease and asthma be tested for AATD. The development of inexpensive and simple genetic testing will help to meet this goal. Methods Primers and synthetic SERPINA1 gene fragments (gBlocks) were designed for 5 AATD-associated variants. PCR was run on a CFX96 Thermal Cycler with High Resolution Melting (HRM) capacity and data analyzed using the supplied HRM-analysis software. Genomic DNA from individuals (n = 86) genotyped for the S and Z variants were used for validation. HRM-analysis was performed on 3 additional samples with low alpha-1-antitrypsin levels inconsistent with the genotype determined in our clinical laboratory. Results Unique normalized melt curve and difference curve patterns were identified for the AAT variants Z, S, I, F, and MMalton using gBlocks. Similar curve shapes were seen when these primers were used to analyze the gDNA samples. HRM identified the genotypes of the gDNA correctly with 100% concordance. The curve shapes of some samples did not match the melting patterns of the targeted variant. Sequencing was used to identify the variants, including rare AATD variants c.1108_1115delinsAAAAACA (p.Glu370Lysfs*31) and c.1130dup (p.Leu377fs). Conclusion We developed a rapid and inexpensive HRM-analysis method for genotyping of Z, S, MMalton, I, and F variants that was also capable of detecting other variants.
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Pencakowski, Bartosz, Monika Bielecka, Marta Stafiniak, Klemens Jakubowski, and Adam Matkowski. "Phylogenetics Study of SalviaL. spp. Collections from the Botanical Garden of Medicinal Plants of Wroclaw Medical University." Biodiversity Information Science and Standards 3 (June 18, 2019). http://dx.doi.org/10.3897/biss.3.36424.
Full textAbstract:
Systematics of genus Salvia L. is still a field of discussion in taxonomic society. To this day, the position of certain genera from Lamiaceae Martinov. family are considered as autonomic genera or incorporated in the genus Salvia L. as a subgenera (i.e., Perovskia Kar., Rosmarinus L.). Moreover, some species are distinguished only by low-level differences in morphological traits and their geographic occurrence (Drew et al. 2017). In this research, we focused on a molecular analysis of morphologically similar Salvia L. species with special attention paid to Salvia glutinosa L. and Salvia nubicola Wall. ex Sweet. All samples were collected from the Botanical Garden of Medicinal Plants of Wroclaw Medical University (http://www.obrl.umed.wroc.pl/index.html) and the Herbarium of Natural History Museum, Wroclaw University (http://www.muzeum-przyrodnicze.uni.wroc.pl/index.php). Several DNA barcodes, including matK, rbcLa, ITS2 genes, and psbA-trnH intergenic spacer, were used in maximal likelihood and Bayesian inference analyses. All sequences were amplified with Q5 High Fidelity DNA Polymerase (https://www.neb.com/) and universal primers. Amplicons were then sequenced by Sanger sequencing and analysed using the BLAST algorithm (Altschul et al. 1990). Subsequently, sequences were aligned using MAFFT v 7.409 (Katoh et al. 2005) software; poorly aligned sites were objectively eliminated with Gblocks v.0.91b (Talavera and Castresana 2007). ITS2 regions were extracted with ITSx (Bengtsson-Palme et al. 2013) software implemented on PlutoF web workbench (Abarenkov et al. 2010) to obtain ITS2 sequences without 5.8S and 26S fragments on both ends. Substitution models were identified utilizing jModelTest 2 software (Posada 2008) and basing on Bayesian information criterion (BIC) appropriate models were applied for further calculations. For maximum likelihood analyses and Bayesian inference, we implemented RAxML ver. 8.2.10 (Stamatakis et al. 2008) and MrBayes ver. 3.2.2 (Ronquist et al. 2012) respectively. For the reconstruction of evolutionary relationship between study species, different DNA barcode combinations were applied (e.g., matK+ITS2+psbA-trnH and rbcLa+matK+ psbA-trnH). Consensus trees were compared and analysed using TreeGraph ver. 2.14 (Stöver and Müller 2010).
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Agarwal, Arati, J. Paul Cunningham, Isabel Valenzuela, and Mark J. Blacket. "A diagnostic LAMP assay for the destructive grapevine insect pest, phylloxera (Daktulosphaira vitifoliae)." Scientific Reports 10, no. 1 (December 2020). http://dx.doi.org/10.1038/s41598-020-77928-9.
Full textAbstract:
AbstractGrape phylloxera (Daktulosphaira vitifoliae) is a destructive insect pest of grapevines that is highly invasive worldwide, despite strict biosecurity containment measures in place at farm and regional levels. Current phylloxera identification by visual inspection and laboratory-based molecular methods is time-consuming and costly. More rapid and cost-effective methods for identification of this pest would benefit industry, growers, and biosecurity services. Loop mediated isothermal amplification (LAMP) is a new portable technology available for rapid and accurate in-field molecular diagnostics. This study outlines the development of a new LAMP assay to enable the identification of phylloxera specimens. New LAMP primers were developed to specifically amplify phylloxera mitochondrial DNA (5′-COI), which we have shown is effective as a DNA barcode for identification of phylloxera, using LAMP technology. Positive LAMP reactions, containing phylloxera DNA, amplified in less than twelve minutes with an anneal derivative temperature of approximately 79 °C to 80 °C compared to a newly designed synthetic DNA (gBlock) fragment which had an anneal derivative temperature of 82 °C. No LAMP amplification was detected in any of the non-target species tested, i.e. no false-positive identification resulted for these species. We also successfully optimised a non-destructive DNA extraction procedure, HotSHOT “HS6”, for use in the field on phylloxera adults, nymphs and eggs, to retain physical specimens. DNA extracted using this method was also suitable for species and genotype molecular identification methods, such as DNA barcoding, qPCR and microsatellite genotyping. The new LAMP assay provides a novel visual molecular tool for accurate diagnostics of phylloxera in the laboratory and field.