An investigation into gene copy number determination in transgenic yeast; The importance of selecting a reliable real-time PCR standard

Shirvani, Roghayeh; Barshan-Tashnizi, Mohammad; Shahali, Maryam

doi:10.1016/j.biologicals.2020.04.001

Cited by 8 publications

(7 citation statements)

References 26 publications

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“…PCR conditions included denaturation at 95 • C, 3 min, followed by 30 cycles of 94 • C, 30 s, 56 • C, 30 s and 72 • C, 30 s and a final extension at 72 • C for 5 min, with the signals collected at the annealing step in each cycle. Analysis of transgene copy number in the pooled gDNAs from LE involved comparison of the qPCR signals obtained using a 10-fold serial dilution series of known quantities of the 24 nt six-stop codon transgene in 235 bp (5 integration PCR fragment) ligated-pCR4-TOPO plasmid DNA (size 4191 bp) to establish a standard curve [68]. The standard curve was based on a linear regression generated using logarithm-10-transformed initial DNA input as the dependent variable and the Ct number of the qPCR signal as the independent variable (Ct values in each dilution were measured in triplicate).…”

Section: Real-time Qpcr To Estimate Transgene Copy Numbersmentioning

confidence: 99%

RNA-Guided AsCas12a- and SpCas9-Catalyzed Knockout and Homology Directed Repair of the Omega-1 Locus of the Human Blood Fluke, Schistosoma mansoni

Ittiprasert

Chatupheeraphat

Mann

et al. 2022

IJMS

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The efficiency of the RNA-guided AsCas12a nuclease of Acidaminococcus sp. was compared with SpCas9 from Streptococcus pyogenes, for functional genomics in Schistosoma mansoni. We deployed optimized conditions for the ratio of guide RNAs to the nuclease, donor templates, and electroporation parameters, to target a key schistosome enzyme termed omega-1. Programmed cleavages catalyzed by Cas12a and Cas9 resulted in staggered- and blunt-ended strand breaks, respectively. AsCas12a was more efficient than SpCas9 for gene knockout, as determined by TIDE analysis. CRISPResso2 analysis confirmed that most mutations were deletions. Knockout efficiency of both nucleases markedly increased in the presence of single-stranded oligodeoxynucleotide (ssODN) template. With AsCas12a, ssODNs representative of both the non-CRISPR target (NT) and target (T) strands were tested, resulting in KO efficiencies of 15.67, 28.71, and 21.43% in the SpCas9 plus ssODN, AsCas12a plus NT-ssODN, and AsCas12a plus T-ssODN groups, respectively. Trans-cleavage against the ssODNs by activated AsCas12a was not apparent in vitro. SpCas9 catalyzed more precise transgene insertion, with knock-in efficiencies of 17.07% for the KI_Cas9 group, 14.58% for KI_Cas12a-NT-ssODN, and 12.37% for KI_Cas12a-T-ssODN. Although AsCas12a induced fewer mutations per genome than SpCas9, the phenotypic impact on transcription and expression of omega-1 was similar for both nucleases.

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Section: Real-time Qpcr To Estimate Transgene Copy Numbersmentioning

confidence: 99%

RNA-Guided AsCas12a- and SpCas9-Catalyzed Knockout and Homology Directed Repair of the Omega-1 Locus of the Human Blood Fluke, Schistosoma mansoni

Ittiprasert

Chatupheeraphat

Mann

et al. 2022

IJMS

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“…Moreover, this yeast offers a very strong methanol-inducible alcohol oxidase (AOX1) promoter which regulate the productivity of its gene interest [15]. The integration of the gene interest to the AOX1 P. pastoris chromosome via homologous recombination generates the stable clones which can be used in high scale fermentation process over generations [16]. Electron microscopy revealed that the pr-ME recombinant protein was seen as VLP as we predicted (data not shown).…”

Section: Discussionmentioning

confidence: 66%

Genetic Stability Test of Pichia pastoris Carrying Integrated Pre-membrane Envelope Gene of DENV3 Indonesian Strain

Sulfianti¹,

Gill²,

Nurhasanah³

et al. 2023

Proceedings of the 1st International Conference for Health Research – BRIN (ICHR 2022)

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In recent years, the methylotrophic Pichia pastoris has become an increasingly famous host for recombinant protein expressions. As a eukaryote, this yeast offers several advantages, including simplicity of genetic manipulation, stable expression, and low-cost scalable fermentation techniques. Previous study has confirmed the insertion of Dengue Virus Serotype 3 (DENV3) pre-Membrane Envelope (pr-M/E) gene in the recombinant P. pastoris X33 generated in our laboratory. The study has also confirmed the strain's ability to express the protein.In this study, we are reporting the genetic stability of the recombinant strain, confirming the steady expression of the heterologous protein in subsequent generations. The genetic stability test was performed by PCR and DNA sequencing on the recombinant P. pastoris cultured in non-baffled shake flasks. Generation time was estimated based on the yeast growth curve and calculated using a previously published formula. According to the growth curve, the generation time of this correlates recombinant yeast is four hours. It differs from the wild type, which took 4.3 h to complete. PCR of target gene performed at generation 1, 18, 39, 56, 81, and 100 revealed two DNA bands which indicating the presence of full AOX1 gene (2.2 kb) and AOX1 promoter plus pr-M/E gene (2 kb). In addition, sequencing of the PCR products show only minor variation, which might have been genuine or a result of PCR or sequencing errors. However, because the amino acid sequences of generation 100 differed from neither the RefSeq nor the original plasmid, we predict that our recombinant P. pastoris stably contains the gene of interest.

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“…7E ). 42,43 Compared with the LiCl or electroporation method ( Table 2 ), the suggested magneto-transformation system achieved an efficient transformation and resulted in sufficient numbers of transformants. Although it is known that the expression of the geneticin resistance gene requires at least two hours, 44 a sufficient number of transformants with geneticin resistance were obtained after 24 hours of the transformation.…”

Section: Resultsmentioning

confidence: 98%

“…This situation might be attributed to occur multi-copy integration of GFP gene into the genomes of yeast cells forming 4 th colony (Fig. 7 E) 42,43 . Comparing with LiCl or electroporation methods (Table 2), the suggested magnetotransformation system achieved an efficient transformation and resulted in sufficient numbers of transformants.…”

Section: Magneto-transformationmentioning

confidence: 99%

Magnetic nanoparticle mediated-gene delivery for simpler and more effective transformation of Pichia pastoris

et al. 2021

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An investigation into gene copy number determination in transgenic yeast; The importance of selecting a reliable real-time PCR standard

Cited by 8 publications

References 26 publications

RNA-Guided AsCas12a- and SpCas9-Catalyzed Knockout and Homology Directed Repair of the Omega-1 Locus of the Human Blood Fluke, Schistosoma mansoni

RNA-Guided AsCas12a- and SpCas9-Catalyzed Knockout and Homology Directed Repair of the Omega-1 Locus of the Human Blood Fluke, Schistosoma mansoni

Genetic Stability Test of Pichia pastoris Carrying Integrated Pre-membrane Envelope Gene of DENV3 Indonesian Strain

Magnetic nanoparticle mediated-gene delivery for simpler and more effective transformation of Pichia pastoris

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