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Improved Gene Targeting through Cell Cycle Synchronization.

Tsakraklides V, Brevnova E, Stephanopoulos G, Shaw AJ - PLoS ONE (2015)

Bottom Line: Gene targeting is a challenge in organisms where non-homologous end-joining is the predominant form of recombination.Using hydroxyurea-mediated cell cycle arrest, we obtained improved gene targeting rates in Yarrowia lipolytica, Arxula adeninivorans, Saccharomyces cerevisiae, Kluyveromyces lactis and Pichia pastoris demonstrating the broad applicability of the method.Hydroxyurea treatment enriches for S-phase cells that are active in homologous recombination and enables previously unattainable genomic modifications.

View Article: PubMed Central - PubMed

Affiliation: Total New Energies, Emeryville, California, United States of America.

ABSTRACT
Gene targeting is a challenge in organisms where non-homologous end-joining is the predominant form of recombination. We show that cell division cycle synchronization can be applied to significantly increase the rate of homologous recombination during transformation. Using hydroxyurea-mediated cell cycle arrest, we obtained improved gene targeting rates in Yarrowia lipolytica, Arxula adeninivorans, Saccharomyces cerevisiae, Kluyveromyces lactis and Pichia pastoris demonstrating the broad applicability of the method. Hydroxyurea treatment enriches for S-phase cells that are active in homologous recombination and enables previously unattainable genomic modifications.

No MeSH data available.


Related in: MedlinePlus

Method for increased gene targeting.Cells are grown in the presence of hydroxyurea to induce cell cycle arrest in S-phase with high HR activity (a). Y. lipolytica YB-392 cells untreated or arrested at the large-budded stage are shown. HU-arrested cells are transformed with an antibiotic resistance cassette bearing the marker flanked by short regions of homology to the promoter and terminator of the target gene (b). Homologous recombination between the cassette and genomic DNA leads to replacement of the target gene with the marker (c). Antibiotic-resistant colonies are screened by PCR to distinguish between random and targeted integration using primer sets specific to each integration outcome (d).
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pone.0133434.g001: Method for increased gene targeting.Cells are grown in the presence of hydroxyurea to induce cell cycle arrest in S-phase with high HR activity (a). Y. lipolytica YB-392 cells untreated or arrested at the large-budded stage are shown. HU-arrested cells are transformed with an antibiotic resistance cassette bearing the marker flanked by short regions of homology to the promoter and terminator of the target gene (b). Homologous recombination between the cassette and genomic DNA leads to replacement of the target gene with the marker (c). Antibiotic-resistant colonies are screened by PCR to distinguish between random and targeted integration using primer sets specific to each integration outcome (d).

Mentions: To test the effect of S-phase arrest on targeted integration in Y. lipolytica, actively dividing cultures of YB-392 were cultivated in the presence or absence of HU prior to transformation. Cell cycle arrest was confirmed by microscopy. An asynchronous Y. lipolytica culture presents a mixed morphology with cells at the unbudded, small-budded and large-budded stage indicating an actively dividing population. HU-treated cells are arrested at the large-budded stage (Fig 1a). Each culture was then carried through the same conventional transformation protocol to introduce a gene deletion cassette encoding a selectable marker flanked by short (37–50 bp) sequences homologous to upstream and downstream regions of the gene of interest (Fig 1b and 1c). Colonies were screened by phenotype or PCR to determine whether marker integration was random or targeted (Fig 1d). HU produced a dramatic shift in favor of targeted integration (Table 1). In agreement with our past experience with this strain, untreated YB-392 transformants contained only randomly integrated marker, rendering gene deletion unattainable. When cells were treated with HU prior to transformation, gene targeting was possible at rates that enable quick and reliable generation of knockouts. To demonstrate the reproducibility of HR stimulation, the deletion of YALI0D17534 which had the lowest level of targeting was performed three times. HU-treated cells showed targeting efficiencies of 4%, 9% and 5% whereas no targeted integration was observed without treatment in 240 colonies screened across the three experiments. We have used this method to delete 15 genes in YB-392. We have observed gene-to-gene variation from 4–96% in the efficiency of gene targeting that may be related to the local chromatin structure or phenotype of deletion (data not shown).


Improved Gene Targeting through Cell Cycle Synchronization.

Tsakraklides V, Brevnova E, Stephanopoulos G, Shaw AJ - PLoS ONE (2015)

Method for increased gene targeting.Cells are grown in the presence of hydroxyurea to induce cell cycle arrest in S-phase with high HR activity (a). Y. lipolytica YB-392 cells untreated or arrested at the large-budded stage are shown. HU-arrested cells are transformed with an antibiotic resistance cassette bearing the marker flanked by short regions of homology to the promoter and terminator of the target gene (b). Homologous recombination between the cassette and genomic DNA leads to replacement of the target gene with the marker (c). Antibiotic-resistant colonies are screened by PCR to distinguish between random and targeted integration using primer sets specific to each integration outcome (d).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4507847&req=5

pone.0133434.g001: Method for increased gene targeting.Cells are grown in the presence of hydroxyurea to induce cell cycle arrest in S-phase with high HR activity (a). Y. lipolytica YB-392 cells untreated or arrested at the large-budded stage are shown. HU-arrested cells are transformed with an antibiotic resistance cassette bearing the marker flanked by short regions of homology to the promoter and terminator of the target gene (b). Homologous recombination between the cassette and genomic DNA leads to replacement of the target gene with the marker (c). Antibiotic-resistant colonies are screened by PCR to distinguish between random and targeted integration using primer sets specific to each integration outcome (d).
Mentions: To test the effect of S-phase arrest on targeted integration in Y. lipolytica, actively dividing cultures of YB-392 were cultivated in the presence or absence of HU prior to transformation. Cell cycle arrest was confirmed by microscopy. An asynchronous Y. lipolytica culture presents a mixed morphology with cells at the unbudded, small-budded and large-budded stage indicating an actively dividing population. HU-treated cells are arrested at the large-budded stage (Fig 1a). Each culture was then carried through the same conventional transformation protocol to introduce a gene deletion cassette encoding a selectable marker flanked by short (37–50 bp) sequences homologous to upstream and downstream regions of the gene of interest (Fig 1b and 1c). Colonies were screened by phenotype or PCR to determine whether marker integration was random or targeted (Fig 1d). HU produced a dramatic shift in favor of targeted integration (Table 1). In agreement with our past experience with this strain, untreated YB-392 transformants contained only randomly integrated marker, rendering gene deletion unattainable. When cells were treated with HU prior to transformation, gene targeting was possible at rates that enable quick and reliable generation of knockouts. To demonstrate the reproducibility of HR stimulation, the deletion of YALI0D17534 which had the lowest level of targeting was performed three times. HU-treated cells showed targeting efficiencies of 4%, 9% and 5% whereas no targeted integration was observed without treatment in 240 colonies screened across the three experiments. We have used this method to delete 15 genes in YB-392. We have observed gene-to-gene variation from 4–96% in the efficiency of gene targeting that may be related to the local chromatin structure or phenotype of deletion (data not shown).

Bottom Line: Gene targeting is a challenge in organisms where non-homologous end-joining is the predominant form of recombination.Using hydroxyurea-mediated cell cycle arrest, we obtained improved gene targeting rates in Yarrowia lipolytica, Arxula adeninivorans, Saccharomyces cerevisiae, Kluyveromyces lactis and Pichia pastoris demonstrating the broad applicability of the method.Hydroxyurea treatment enriches for S-phase cells that are active in homologous recombination and enables previously unattainable genomic modifications.

View Article: PubMed Central - PubMed

Affiliation: Total New Energies, Emeryville, California, United States of America.

ABSTRACT
Gene targeting is a challenge in organisms where non-homologous end-joining is the predominant form of recombination. We show that cell division cycle synchronization can be applied to significantly increase the rate of homologous recombination during transformation. Using hydroxyurea-mediated cell cycle arrest, we obtained improved gene targeting rates in Yarrowia lipolytica, Arxula adeninivorans, Saccharomyces cerevisiae, Kluyveromyces lactis and Pichia pastoris demonstrating the broad applicability of the method. Hydroxyurea treatment enriches for S-phase cells that are active in homologous recombination and enables previously unattainable genomic modifications.

No MeSH data available.


Related in: MedlinePlus