Showing papers on "Cloning published in 2018"
TL;DR: The Gateway recombinatorial cloning system was developed for cloning multiple DNA fragments in parallel in a standardized manner using the same enzymes to robustly clone many different fragments of variable size in parallel reactions.
Abstract: The Gateway recombinatorial cloning system was developed for cloning multiple DNA fragments in parallel (e.g., in 96-well formats) in a standardized manner using the same enzymes. Gateway cloning is based on the highly specific integration and excision reactions of bacteriophage λ into and out of the Escherichia coli genome. Because the sites of recombination ("att" sites) are much longer (25-242 bp) than restriction sites, they are extremely unlikely to occur by chance in DNA fragments. Therefore, the same recombination enzyme can be used to robustly clone many different fragments of variable size in parallel reactions.
65 citations
TL;DR: This work has further streamlined DiPaC by reducing cloning time and reagent costs by utilizing T4 DNA polymerase (sequence- and ligation-independent cloning, SLIC) for gene cluster capture and depicts a time and cost-efficient method for simple capture and expression of new natural product pathways.
Abstract: The need for new pharmacological lead structures, especially against drug resistances, has led to a surge in natural product research and discovery. New biosynthetic gene cluster capturing methods to efficiently clone and heterologously express natural product pathways have thus been developed. Direct pathway cloning (DiPaC) is an emerging synthetic biology strategy that utilizes long-amplification PCR and HiFi DNA assembly for the capture and expression of natural product biosynthetic gene clusters. Here, we have further streamlined DiPaC by reducing cloning time and reagent costs by utilizing T4 DNA polymerase (sequence- and ligation-independent cloning, SLIC) for gene cluster capture. As a proof of principle, the majority of the cyanobacterial hapalosin gene cluster was cloned as a single piece (23 kb PCR product) using this approach, and predicted transcriptional terminators were removed by simultaneous pathway refactoring, leading to successful heterologous expression. The complementation of DiPaC w...
47 citations
TL;DR: It is revealed that the monocot-specific GPAT3 protein plays an important role in male fertility in maize, and ZmMs33 and mutants in this gene may have value in maize male-sterile line breeding and hybrid seed production.
Abstract: Map-based cloning of maize ms33 gene showed that ZmMs33 encodes a sn-2 glycerol-3-phosphate acyltransferase, the ortholog of rice OsGPAT3, and it is essential for male fertility in maize. Genetic male sterility has been widely studied for its biological significance and commercial value in hybrid seed production. Although many male-sterile mutants have been identified in maize (Zea mays L.), it is likely that most genes that cause male sterility are unknown. Here, we report a recessive genetic male-sterile mutant, male sterility33 (ms33), which displays small, pale yellow anthers, and complete male sterility. Using a map-based cloning approach, maize GRMZM2G070304 was identified as the ms33 gene (ZmMs33). ZmMs33 encodes a novel sn-2 glycerol-3-phosphate acyltransferase (GPAT) in maize. A functional complementation experiment showed that GRMZM2G070304 can rescue the male-sterile phenotype of the ms33-6029 mutant. GRMZM2G070304 was further confirmed to be the ms33 gene via targeted knockouts induced by the clustered regularly interspersed short palindromic repeats (CRISPR)/Cas9 system. ZmMs33 is preferentially expressed in the immature anther from the quartet to early-vacuolate microspore stages and in root tissues at the fifth leaf growth stage. Phylogenetic analysis indicated that ZmMs33 and OsGPAT3 are evolutionarily conserved for anther and pollen development in monocot species. This study reveals that the monocot-specific GPAT3 protein plays an important role in male fertility in maize, and ZmMs33 and mutants in this gene may have value in maize male-sterile line breeding and hybrid seed production.
45 citations
TL;DR: A Leucine Rich Repeat family gene (LOC_Os04g02520) was identified as the most possible candidate of Bph33, a new resistance gene that exhibited continuous and stable resistance from seedling stage to tillering stage, showing both antixenosis and antibiosis effects on BPH.
Abstract: Host-plant resistance is the most desirable and economic way to overcome BPH damage to rice. As single-gene resistance is easily lost due to the evolution of new BPH biotypes, it is urgent to explore and identify new BPH resistance genes. In this study, using F2:3 populations and near-isogenic lines (NILs) derived from crosses between two BPH-resistant Sri Lankan rice cultivars (KOLAYAL and POLIYAL) and a BPH-susceptible cultivar 9311, a new resistance gene Bph33 was fine mapped to a 60-kb region ranging 0.91–0.97 Mb on the short arm of chromosome 4 (4S), which was at least 4 Mb distant from those genes/QTLs (Bph12, Bph15, Bph3, Bph20, QBph4 and QBph4.2) reported before. Seven genes were predicted in this region. Based on sequence and expression analyses, a Leucine Rich Repeat (LRR) family gene (LOC_Os04g02520) was identified as the most possible candidate of Bph33. The gene exhibited continuous and stable resistance from seedling stage to tillering stage, showing both antixenosis and antibiosis effects on BPH. The results of this study will facilitate map-based cloning and marker-assisted selection of the gene.
32 citations
TL;DR: It was proved in recent studies on pig cloning that the effectiveness of piezo-driven microinjection of ear-derived fibroblast karyoplasts measured by the percentage of oocytes preserving vitality after cell nuclei transplantation operation did not differ significantly from the survival rate (viability) of clonal cybrids reconstituted by an electrofusion method.
Abstract: The introduction of cell nuclei into enucleated recipient cells, beyond enucleation, is the most sig- nificant stage of somatic cloning procedure. Microsurgical transfer of somatic nuclei can be an alternative method of clonal nuclear-cytoplasmic hybrid reconstruction towards cell fusion induced in the electric field, not only from the aspect of molecular mechanisms of nuclear chromatin rearrangement, advantageously influencing epigenetic reprogramming and structural remodelling of exogenous genetic material, but also because it was proved in recent studies on pig cloning that the effectiveness of piezo-driven microinjection of ear-derived fibroblast karyoplasts measured by the percentage of oocytes preserving vitality after cell nuclei transplantation operation did not differ significantly from the survival rate (viability) of clonal cybrids reconstituted by an electrofusion method. The intraooplasmic injection system of karyoplasts prepared from cells at G0/G1 or G2/M stages of cell cycle could also increase considerably the total efficiency of somatic cloning technique in pigs and other mammal species.
24 citations
TL;DR: Methylome and transcriptome analyses of pig iPSCs and associated cloned embryos revealed that aberrant silencing of imprinted genes, in particular the retrotransposon-derived RTL1 gene, is the principal epigenetic cause of pregnancy failure.
Abstract: Substantial rates of fetal loss plague all in vitro procedures involving embryo manipulations, including human-assisted reproduction, and are especially problematic for mammalian cloning where over 90% of reconstructed nuclear transfer embryos are typically lost during pregnancy. However, the epigenetic mechanism of these pregnancy failures has not been well described. Here we performed methylome and transcriptome analyses of pig induced pluripotent stem cells and associated cloned embryos, and revealed that aberrant silencing of imprinted genes, in particular the retrotransposon-derived RTL1 gene, is the principal epigenetic cause of pregnancy failure. Remarkably, restoration of RTL1 expression in pig induced pluripotent stem cells rescued fetal loss. Furthermore, in other mammals, including humans, low RTL1 levels appear to be the main epigenetic cause of pregnancy failure.
23 citations
TL;DR: Evidence is provided that the in vivo cloning of E. coli is independent of both RecA and RecET recombinase, but is dependent on XthA, a 3’ to 5’ exonuclease, and the iVEC activity is reduced by deletion of the C-terminal domain of DNA polymerase I (PolA).
Abstract: Escherichia coli has an ability to assemble DNA fragments with homologous overlapping sequences of 15-40 bp at each end. Several modified protocols have already been reported to improve this simple and useful DNA-cloning technology. However, the molecular mechanism by which E. coli accomplishes such cloning is still unknown. In this study, we provide evidence that the in vivo cloning of E. coli is independent of both RecA and RecET recombinase, but is dependent on XthA, a 3’ to 5’ exonuclease. Here, in vivo cloning of E. coli by XthA is referred to as iVEC (in vivo E. coli cloning). Next, we show that the iVEC activity is reduced by deletion of the C-terminal domain of DNA polymerase I (PolA). Collectively, these results suggest the following mechanism of iVEC. First, XthA resects the 3′ ends of linear DNA fragments that are introduced into E. coli cells, resulting in exposure of the single-stranded 5′ overhangs. Then, the complementary single-stranded DNA ends hybridize each other, and gaps are filled by DNA polymerase I. Elucidation of the iVEC mechanism at the molecular level would further advance the development of in vivo DNA-cloning technology. Already we have successfully demonstrated multiple-fragment assembly of up to seven fragments in combination with an effortless transformation procedure using a modified host strain for iVEC. Importance Cloning of a DNA fragment into a vector is one of the fundamental techniques in recombinant DNA technology. Recently, in vitro recombination of DNA fragments effectively joins multiple DNA fragments in place of the canonical method. Interestingly, E. coli can take up linear double-stranded vectors, insert DNA fragments and assemble them in vivo. The in vivo cloning have realized a high level of usability comparable to that by in vitro recombination reaction, since now it is only necessary to introduce PCR products into E. coli for the in vivo cloning. However, the mechanism of in vivo cloning is highly controversial. Here we clarified the fundamental mechanism underlying in vivo cloning of E. coli and also constructed an E. coli strain that was optimized for in vivo cloning.
21 citations
TL;DR: This approach provides a fast and easy-to-implement strategy for engineering metabolic pathway flux for development of industrially relevant strains, as well as investigation of fundamental biological questions.
Abstract: Optimization of metabolic flux is a difficult and time-consuming process that often involves changing the expression levels of multiple genes simultaneously. While some pathways have a known rate limiting step, more complex metabolic networks can require a trial-and-error approach of tuning the expression of multiple genes to achieve a desired distribution of metabolic resources. Here we present an efficient method for generating expression diversity on a combinatorial scale using CRISPR interference. We use a modified native Escherichia coli Type I-E CRISPR-Cas system and an iterative cloning strategy for construction of guide RNA arrays. This approach allowed us to build a combinatorial gene expression library three orders of magnitude larger than previous studies. In less than 1 month, we generated ∼12,000 combinatorial gene expression variants that target six different genes and screened these variants for increased malonyl-CoA flux and 3-hydroxypropionate (3HP) production. We were able to identify a set of variants that exhibited a significant increase in malonyl-CoA flux and up to a 98% increase in 3HP production. This approach provides a fast and easy-to-implement strategy for engineering metabolic pathway flux for development of industrially relevant strains, as well as investigation of fundamental biological questions.
20 citations
01 Oct 2018
TL;DR: Newly developed pAL vectors now reduce basal expression to levels where coding sequences for even the most stressful proteins can be maintained and induced, and asymmetric ligation allows simple and efficient cloning of individual coding sequences or simultaneous cloning of two or three coding sequences from a single pAL vector.
Abstract: Inducible T7 expression systems are capable of producing a wide range of proteins in E. coli. Improvements over common practice include: (1) preventing unintended induction by establishing and maintaining expression strains in non-inducing growth media composed entirely of purified components instead of complex growth media that may variably induce target proteins on approach to saturation; and (2) expressing many target proteins in parallel by convenient and productive auto-induction in BL21(DE3) and other suitable hosts, instead of IPTG induction. From the earliest days, basal expression prevented establishment of inducible strains for producing proteins that are stressful to the host. Newly developed pAL vectors now reduce basal expression to levels where coding sequences for even the most stressful proteins can be maintained and induced. Asymmetric ligation allows simple and efficient cloning of individual coding sequences or simultaneous cloning of two or three coding sequences for co-expression from a single pAL vector. © 2018 by John Wiley & Sons, Inc.
17 citations
TL;DR: The results suggest that this satellite I region could be used as an epigenetic biomarker for predicting offspring viability by evaluating DNA methylation patterns of this satellite region in the donor cell genome or embryo biopsies.
17 citations
TL;DR: A versatile series of mycobacterial vectors for gene deletion, complementation and protein production and purification, compatible with fragment exchange (FX) cloning, and used to delete twenty genes encoding for potential drug efflux pumps in M. smegmatis.
Abstract: Molecular research on mycobacteria relies on a multitude of tools for the genetic manipulation of these clinically important bacteria. However, a uniform set of vectors allowing for standardized cloning procedures is not available. Here, we developed a versatile series of mycobacterial vectors for gene deletion, complementation and protein production and purification. The vectors are compatible with fragment exchange (FX) cloning, a recently developed high-throughput cloning principle taking advantage of the type IIS restriction enzyme SapI and its capacity to generate sticky trinucleotide ends outside of its recognition sequence. FX cloning allows for the efficient cloning into an entry vector and the facile transfer of the sequenced insert into a variety of destination vectors. We generated a set of mycobacterial expression vectors spanning a wide range of expression strengths, tagging variants and selection markers to rapidly screen for the optimal expression construct in order to purify membrane proteins from the model organism Mycobacterium smegmatis. Further, we generated a series of suicide vectors containing two counterselection markers and used them to delete twenty genes encoding for potential drug efflux pumps in M. smegmatis. The vectors will further facilitate genetic and biochemical research on various mycobacterial species.
TL;DR: It is suggested that optimum exposure to CI994 after activation elevates the level of histone acetylation and subsequently improves the in vitro development of pig SCNT embryos.
Abstract: Epigenetic reprogramming and somatic cell nuclear transfer (SCNT) cloning efficiency were recently enhanced using histone deacetylase inhibitors (HDACis). In this study, we investigated th...
TL;DR: Easy preparation of monoclonal insect cell lines in a serum free environment using co-cultivation with untreated feeder cells and antibiotic selection and expression of a fluorescent reporter and two antimicrobial peptides.
TL;DR: This somatic cell nuclear transfer method involves usage of Hemagglutinating virus of Japan Envelope (HVJ-E), which enables easy manipulation and requires neither additional injection nor genetic manipulation, and thus presents a simple, suitable method for practical use.
Abstract: Somatic cell nuclear transfer (SCNT) provides a unique opportunity to directly produce a cloned animal from a donor cell, and it requires the use of skillful techniques. Additionally, the efficiencies of cloning have remained low since the successful production of cloned animals, especially mice. There have been many attempts to improve the cloning efficiency, and trichostatin A (TSA), a histone deacetylase inhibitor, has been widely used to enhance the efficiency of cloning. Here, we report a dramatically improved cloning method in mice. This somatic cell nuclear transfer method involves usage of Hemagglutinating virus of Japan Envelope (HVJ-E), which enables easy manipulation. Moreover, the treatment using two small molecules, TSA and vitamin C (VC), with deionized bovine serum albumin (dBSA), is highly effective for embryonic development. This approach requires neither additional injection nor genetic manipulation, and thus presents a simple, suitable method for practical use. This method could become a technically feasible approach for researchers to produce genetically modified animals from cultured cells. Furthermore, it might be a useful way for the rescue of endangered animals via cloning.
TL;DR: Here, the multiple cloning sites from commonly used expression vectors are modified to create a new suite of cloning plasmids that simplify and speed up cloning procedures in Escherichia coli.
Abstract: Here, we modified the multiple cloning sites from commonly used expression vectors to create a new suite of cloning plasmids that simplify and speed up cloning procedures in Escherichia coli. Each of our standardized plasmids contains two BsaI restriction sites, allowing for highly efficient cloning of genes and bringing their expression under control of either a T7 (pET21a_BsaI, pET28a_BsaI, and pMAL-c5T_BsaI) or T5 promoter (pUR22 and pUR23). Another plasmid in our suite (pTNA_BsaI) allows for generation of large gene libraries containing >108 variants, which can be constitutively expressed in selection experiments using metabolic complementation of auxotrophic E. coli strains. Coupling restriction and ligation with the BsaI restriction enzyme minimizes hands-on time, while the need for only three different primers to clone a target gene into the six different vectors keeps overall cloning costs low.
TL;DR: In recent studies on pig cloning it was proved that the effectiveness of piezo-driven microinjection of ear-derived fibroblast karyoplasts measured by the percentage of oocytes preserving vitality after the cell nuclei transplantation operation did not differ significantly from the survival rate (viability) of clonal cybrids reconstituted by an electrofusion method.
Abstract: The introduction of cell nuclei into enucleated recipient cells, beyond enucleation, is the most sig- nificant stage of somatic cloning procedure. Microsurgical transfer of somatic nuclei can be an alternative method of clonal nuclear-cytoplasmic hybrid reconstruction towards cell fusion induced in the electric field, not only from the aspect of molecular mechanisms of nuclear chromatin rearrangement, advantageously influencing epigenetic reprogramming and structural remodelling of exogenous genetic material but also because in recent studies on pig cloning it was proved that the effectiveness of piezo-driven microinjection of ear-derived fibroblast karyoplasts measured by the percentage of oocytes preserving vitality after the cell nuclei transplantation operation did not differ significantly from the survival rate (viability) of clonal cybrids reconstituted by an electrofusion method. An intraooplasmic injection system of karyoplasts prepared from cells at G0/G1 or G2/M stages of cell cycle could also increase considerably the total efficiency of somatic cloning technique in pigs and in other mammal species.
TL;DR: The reproductive cloning in buffalo in India has been started using a simplified somatic cell nuclear transfer technique named handmade cloning, and chemical epigenetic modifiers can be used to treat donor somatic cells and reconstructed fused embryos to correct the epigenetic reprogramming to enhance the overall cloning efficiency.
Abstract: The reproductive cloning in buffalo in India has been started using a simplified somatic cell nuclear transfer technique named handmade cloning. Since the birth of first cloned female buffalo in 2009, a number of buffalo clones have been produced in India by utilizing different types of donor cells such as ear cells, embryonic stem cells, semen somatic cells and urine somatic cells. The use of buffalo cloning on a large scale is restricted due to low pregnancy rates and poor calf survival. Considerable attempts have been made to improve the overall buffalo cloning efficiency, particularly by modifying epigenetic reprogramming of cloned embryos. Previous studies have demonstrated that chemical epigenetic modifiers such as trichostatin A and 5-aza-2'-deoxycytidine, m-carboxycinnamic acid bishydroxamide can be used to treat donor somatic cells and reconstructed fused embryos to correct the epigenetic reprogramming to enhance the overall cloning efficiency in terms of live birth rates.
TL;DR: This report shows that molecular cloning plasmids bearing the coding sequence for a 25.4 kDa protein, AmilCP, encoded by a 685 bp gene, that is well expressed in Escherichia coli, render blue-purple colonies and develops and tests a cloning system based on the constitutive expression of the non-toxic Amil CP protein, that once interrupted, the loss of purple color serves to facilitate positive clone selection.
Abstract: Cloning and expression plasmids are the workhorses of modern molecular biology. Despite the pathway paved by synthetic biology, laboratories around the globe still relay on standard cloning techniques using plasmids with reporter proteins for positive clone selection, such as β-galactosidase alpha peptide complementation for blue/white screening or ccdB, which encodes for a toxic DNA gyrase. These reporters, when interrupted, serve as a positive clone detection system. In the present report, we show that molecular cloning plasmids bearing the coding sequence for a 25.4 kDa protein, AmilCP, encoded by a 685 bp gene, that is well expressed in Escherichia coli, render blue-purple colonies. Using this reporter protein, we developed and tested a cloning system based on the constitutive expression of the non-toxic AmilCP protein, that once interrupted, the loss of purple color serves to facilitate positive clone selection. The main advantage of this system is that is less expensive than other systems since media do not contain chromogenic markers such as X-gal, which is both expensive and cumbersome to prepare and use, or inductors such as IPTG. We also designed an inducible expression plasmid suitable for recombinant protein expression that also contains AmilCP cloning selection marker, a feature not commonly found in protein expression plasmids. The use of chromogenic reporters opens an important avenue for its application in other organisms besides E. coli for clone selection or even for mutant selection.
TL;DR: Serial cloning by somatic cell nuclear transfer is a critical tool for the expansion of precious transgenic lines or resetting the lifespan of primary transgenic cells for multiple genetic modifications and methylation patterns were detected in G1 and G2 clones.
Abstract: Serial cloning by somatic cell nuclear transfer (SCNT) is a critical tool for the expansion of precious transgenic lines or resetting the lifespan of primary transgenic cells for multiple genetic modifications. We successfully produced second-generation cloned goats using donor neonatal fibroblasts from first-generation clones. However, our attempts to produce any third-generation clones failed. SCNT efficiency decreased progressively with the clonal generations. The rate of pregnancy loss was significantly greater in recloning groups (P<0.05). While no pregnancy loss was observed during the first round of SCNT, 14 out of 21 pregnancies aborted in the second round of SCNT and all pregnancies aborted in the third round of SCNT. In this retrospective study, we also investigated the expression of 21 developmentally important genes in muscle tissue of cloned (G1) and recloned (G2) offspring. The expression of most of these genes in live clones was found to be largely comparable to naturally reproduced control goats, but fibroblast growth factor 10 (FGF10), methyl CpG binding protein 2 (MECP2) and growth factor receptor bound protein 10 (GRB10) were differentially expressed (P<0.05) in G2 goats compared with G1 and controls. To study the effects of serial cloning on DNA methylation, the methylation pattern of differentially methylated regions in imprinted genes H19 and insulin like growth factor 2 receptor (IGF2R) were also analysed. Aberrant H19 DNA methylation patterns were detected in G1 and G2 clones.
TL;DR: Map‐based cloning revealed that TCM1 encodes a novel chloroplast‐targeted TAC protein in rice, in which a mutation leads to an albino phenotype and malformed chloroplasts before the three‐leaf stage at low temperatures.
Abstract: Transcriptionally active chromosome (TAC) is a component of protein-DNA complexes with RNA polymerase activity, expressed in the plastid. However, the function of rice TAC proteins is still poorly understood. In this paper, we first report the identification of a new rice ( L.) mutant () in the gene encoding TAC. The mutant displayed an albino phenotype and malformed chloroplasts before the three-leaf stage when grown at low temperatures (20°C) and a normal phenotype at higher temperatures (>28°C). Map-based cloning revealed that encodes a novel chloroplast-targeted TAC protein in rice. In addition, the transcript levels of all examined plastid-encoded polymerase (PEP)-dependent genes were clearly downregulated in mutants at low temperatures, although partially recovering levels were obtained at high temperatures, comparable to wild-type plants. Furthermore, the transcripts were ubiquitously expressed in all examined tissues, with high expression levels in green tissues. The data suggest that the rice nuclear-encoded TAC protein TCM1 is essential for proper chloroplast development and maintaining PEP activity under cold stress.
TL;DR: This work focuses on recent BAC cloning techniques of Epstein-Barr virus (EBV) genomes and discusses the possible advantages of a CRISPR/Cas9-mediated strategy comparatively with precedent EBV-BAC cloning strategies.
Abstract: Herpesviruses have relatively large DNA genomes of more than 150 kb that are difficult to clone and sequence. Bacterial artificial chromosome (BAC) cloning of herpesvirus genomes is a powerful technique that greatly facilitates whole viral genome sequencing as well as functional characterization of reconstituted viruses. We describe recently invented technologies for rapid BAC cloning of herpesvirus genomes using CRISPR/Cas9-mediated homology-directed repair. We focus on recent BAC cloning techniques of Epstein-Barr virus (EBV) genomes and discuss the possible advantages of a CRISPR/Cas9-mediated strategy comparatively with precedent EBV-BAC cloning strategies. We also describe the design decisions of this technology as well as possible pitfalls and points to be improved in the future. The obtained EBV-BAC clones are subjected to long-read sequencing analysis to determine complete EBV genome sequence including repetitive regions. Rapid cloning and sequence determination of various EBV strains will greatly contribute to the understanding of their global geographical distribution. This technology can also be used to clone disease-associated EBV strains and test the hypothesis that they have special features that distinguish them from strains that infect asymptomatically.
TL;DR: The isolation of camelderived variable domains of the heavy chain antibody (VHH, also called nanobodies) directed against the coat protein (CP) of Zucchini yellow mosaic virus showed binding not only to recombinant ZYMV-CP but also to native ZyMV, indicating that thesenanobodies can be used in diagnostic tools to detect viral infections.
Abstract: Zakri A.M., Al-Doss A.A., Sack M., Ali A.A., Samara E.M., Ahmed B.S., Amer M.A., Abdalla O.A., Al-Salehd M.A. (2018): Cloning and characterisation of nanobodies against the coat protein of Zucchini yellow mosaic virus. Plant Protect. Sci., 54: 215–221. Zucchini yellow mosaic virus (ZYMV), in the family Potyviridae, causes an economically important disease. Antibodies are valuable reagents for diagnostic assays to rapidly detect viral infection. Here, we report the isolation of camelderived variable domains of the heavy chain antibody (VHH, also called nanobodies) directed against the coat protein (CP) of ZYMV. Several nanobodies that specifically recognise ZYMV-CP were identified. The isolated nanobodies showed binding not only to recombinant ZYMV-CP but also to native ZYMV, indicating that these nanobodies can be used in diagnostic tools to detect viral infections.
TL;DR: It is demonstrated that in vitro development of clones is not hindered by pharmacologically treating either the donor cells or the embryos themselves with CPI, PS48, or the combination of these drugs and early embryos have a low proportion of mitochondria which have high membrane potential and treatment with these pharmaceuticals does not further alter the mitochondrial function in early embryos.
Abstract: Somatic cell nuclear transfer is a valuable technique for the generation of genetically engineered animals, however, the efficiency of cloning in mammalian species is low (1-3%). Differentiated somatic cells commonly used in nuclear transfer utilize the tricarboxylic acid cycle and cellular respiration for energy production. Comparatively the metabolism of somatic cells contrasts that of the cells within the early embryos which predominately use glycolysis. Early embryos (prior to implantation) are evidenced to exhibit characteristics of a Warburg Effect (WE)-like metabolism. We hypothesized that pharmacologically driven fibroblast cells can become more blastomere-like and result in improved in vitro embryonic development after SCNT. The goals were to determine if subsequent in vitro embryo development is impacted by (1) cloning pharmacologically treated donor cells pushed to have a WE-like metabolism or (2) culturing non-treated donor clones with pharmaceuticals used to push a WE-like metabolism. Additionally, we investigated early gestational survival of the donor-treated clone embryos. Here we demonstrate that in vitro development of clones is not hindered by pharmacologically treating either the donor cells or the embryos themselves with CPI, PS48, or the combination of these drugs. Furthermore, these experiments demonstrate that early embryos (or at least in vitro produced embryos) have a low proportion of mitochondria which have high membrane potential and treatment with these pharmaceuticals does not further alter the mitochondrial function in early embryos. Lastly, we show that survival in early gestation was not different between clones from pharmacologically induced WE-like donor cells and controls.
TL;DR: This finding strongly suggests that the translational product of the ldhL2 gene does not exhibit L-LDH activity in vivo, and there is a demand to incorporate acid-resistance for effective LA production.
Abstract: Lactic acid (LA) has been used for wide range of food processing and industrial applications, for example, as a raw material of biodegradable plastics, poly(lactic acid)s (PLAs). Thus, there is a demand to incorporate acid-resistance for effective LA production. Acid-tolerant lactic acid bacteria (LAB), Lactobacillus acetotolerans HT, has been isolated from rice vinegar (pH 2.9, 6 % acetic acid). In this study, genes for the Dand L-lactate dehydrogenases (D-LDH, L-LDH1, and L-LDH2) of Lb. acetotolerans HT, which constitute the key enzymes in cell growth and lactic acid production, were cloned and identified. Through heterologous expression of LDH genes in Escherichia coli DH5α, recombinant E. coli DH5α harboring the D-LDH (ldhD) or L-LDH1 (ldhL1) genes were found to produce D-LA or L-LA, respectively, whereas the strain harboring the L-LDH2 (ldhL2) gene did not produce L-LA. This finding strongly suggests that the translational product of the ldhL2 gene does not exhibit L-LDH activity in vivo.
TL;DR: The feasibility of using pXST vector to clone PCR products amplified from cassava genome with both TA cloning and blunt-end ligation methods is confirmed and would offer flexible alternatives for gene cloning experiments to researchers.
Abstract: With the rapid development of sequencing technologies, increasing amount of genomic information has been accumulated. To clone genes for further functional studies in large scale, a cheap, fast and efficient cloning vector is desired. A bifunctional vector pXST has been constructed. The pXST vector harbors a XcmI-ccdB-XcmI cassette and restriction site SmaI. Digestion the vector with XcmI generates a single thymidine (T) overhang at 3′ end which facilitates TA cloning, and SmaI gives blunt end that enables the blunt-end ligation. Multiple products with various sizes were amplified from cassava genome by PCR and each PCR fragment was separately cloned into a pXST using TA cloning and blunt-end ligation methods. In general, the TA cloning gave higher transformation efficiency than blunt-end ligation for inserts with all different sizes, and the transformation efficiency significantly decreased with increasing size of inserts. The highest transformation efficiency (8.6 × 106 transformants/μg) was achieved when cloning 517 bp DNA fragment using TA cloning. No significant difference observed in the positive cloning efficiency between two ligation methods and the positive cloning efficiency could reach as high as 100% especially for small inserts (e.g. 517 and 957 base pairs). We describe a simple and general method to construct a novel pXST vector. We confirm the feasibility of using pXST vector to clone PCR products amplified from cassava genome with both TA cloning and blunt-end ligation methods. The pXST plasmid has several advantages over many currently available vectors in that (1) it possesses XcmI-ccdB-XcmI cassette and restriction site SmaI, enabling both TA cloning and blunt-end ligation. (2) it allows direct selection of positive recombinant plasmids in Escherichia coli through disruption of the ccdB gene. (3) it improves positive cloning efficiency by introducing the ccdB gene, reducing the possibility of self-ligation from insufficient digested plasmids. (4) it could be used by high performance and cost-effective cloning methods. Therefore, this dual function vector would offer flexible alternatives for gene cloning experiments to researchers.
TL;DR: The generation of pICOz is described, a 1185 bp fully functional high-copy cloning plasmid with an extended multiple cloning site (MCS), to the authors' knowledge, this is the smallest high- copy cloning vector ever described.
Abstract: Minimal plasmids play an essential role in many intermediate steps in molecular biology. They can for example be used to assemble building blocks in synthetic biology or be used as intermediate cloning plasmids that are ideal for PCR-based mutagenesis methods. A small backbone also opens up for additional unique restriction enzyme cloning sites. Here we describe the generation of pICOz, a 1185 bp fully functional high-copy cloning plasmid with an extended multiple cloning site (MCS). To our knowledge, this is the smallest high-copy cloning vector ever described.
Journal Article•
TL;DR: The cloning and expression analysis of XsLEC1 gene provide an important foundation to further study the function and are of practical significance for improving the quality of the X. sorbifolia oil.
Abstract: [Objective] Xanthoceras sorbifolia, belonging to the family Sapindaceae, is one of the most valuable oil trees widely distributed in northern China. Its seeds contain a large amount of oil that is high鄄 quality raw material for food and biodiesel purposes. This study aims to clone Xanthoceras sorbifolia Leafy Cotyledon 1gene (XsLEC1) and analyze its sequence and expression pattern. [Method] XsLEC1 gene was cloned by RT鄄PCR and RACE methods from developing X. sorbifolia embryos. Physicochemical properties and transmembrane regions of the deduced XsLEC1 protein were predicted via Protparam and TMHMM2郾 0 programs, respectively. Subcellular localization and function of XsLEC1 protein were 摇 第 1 期 路蒙蒙等: 文冠果 LEC1 基因的克隆及表达分析 predicted by ProtComp9郾 0 and Motif Scan programs, respectively. Multiple sequence alignment was carried out using BioEdit software and phylogenetic analysis was achieved by MEGA7 software. Expression pattern of XsLEC1 was analyzed by RT鄄PCR and quantitative real鄄time PCR. [Result] XsLEC1 cDNA was 1 035 bp in length, containing a 690 bp ORF ( GenBank accession numbers: MF616360) and encoding a putative protein with 229 amino acids. The deduced amino acid sequence of XsLEC1 contained a conserved B domain of the HAP3 subunit. The B domain contained three 琢鄄helices (琢1,琢2,琢3) and two loops (L1,L2) in the histone fold motif. The predicted XsLEC1 was a stable hydrophilic protein without signal peptide and transmembrane regions. The protein was located in the nucleus and contained many phosphorylation sites. The secondary structure of XsLEC1 protein was mainly consisted of alpha helix and random coil. Phylogenetic analysis showed that XsLEC1 was the closest to DlLEC1, followed by JcLEC1 and PtLEC1. The RT鄄PCR revealed that XsLEC1 was not expressed in roots, stems, leaves, and petals, but highly expressed in the developing embryos. Quantitative real鄄time PCR indicated that XsLEC1 had temporal expression pattern in developing X. sorbifolia embryos. The XsLEC1 expression was higher in early embryo development (33, 40 and 47 days after anthesis) than in late embryo development (54, 61 and 68 days after anthesis) . With the embryo maturation, the XsLEC1 expression was very low at 75 days after anthesis and no transcript was detected at 81 days after anthesis. [Conclusion] The cloning and expression analysis of XsLEC1 gene provide an important foundation to further study the function of XsLEC1 and are of practical significance for improving the quality of the X. sorbifolia oil.
01 Aug 2018
TL;DR: In this article, the basic structure of AP2 gene was unchanged during evolution in diploid species, and three AtAP2 orthologs were cloned from Brassica juncea and B. nigra.
Abstract: The APETALA2/Ethylene-Responsive Factor (AP2/ERF) is one of the largest gene families encoding several plant specific transcription factors. It plays significant roles in growth and development process, biotic and abiotic stresses, and responses to hormones. AP2 is a homeotic gene governing floral meristem specification, floral organ determination and floral homeotic gene expression in Arabidopsis. The basic structure of AP2 gene was unchanged during evolution in diploid species. The present study was undertaken to find whether AP2 has undergone any change in structure or expression pattern during evolution of allopolyploid Brassica juncea. We cloned AP2 orthologs and c-DNAs from B. juncea and B. nigra. B. juncea was found to carry three AtAP2 orthologs. Comparison of BjAP2 genes with AP2 orthologs from progenitor species, B. rapa and B. nigra showed that two of the BjAP2 genes were derived from B. rapa and one from B. nigra. BjAP2 genes have retained its characteristic AP2 domain and miR172 complementary sequences. mRNAs originated from three AP2 orthologs were detected in all the tissues examined, namely, leaf, flower buds and seedling, indicating absence of subfunctionalization of AP2 during polyploid evolution. However, one of the B. rapa copies gave alternatively spliced AP2 transcript which lacked the second exon. Consequently, the splice variant could not be translated into functional AP2 protein. Considering that miR172 suppresses translation of AP2 transcripts, the alternatively spliced transcript could still play important regulatory role by limiting the availability of miR172 molecules to bind to functional AP2 transcripts. qRT-PCR analysis of BjAP2 expression in different accessions of B. juncea with contrasting seed size indicated that BjAP2 is not a major determinant of seed size in mustard.