Adaptation of the AID system for stem cell and transgenic mouse research

Pryzhkova, Marina V.; Xu, Michelle J; Jordan, Philip W.

doi:10.1016/j.scr.2020.102078

Cited by 9 publications

(10 citation statements)

References 79 publications

(124 reference statements)

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“…B6 mESCs used in this study were established and maintained in 2i/LIF medium as described in (Pryzhkova and Jordan, 2016;Pryzhkova et al, 2020b). mESCs were verified to be negative for mycoplasma using the PCR Mycoplasma Test Kit I/C (PromoCell).…”

Section: Mesc Culture and Analysismentioning

confidence: 99%

SMC5/6 is required for replication fork stability and faithful chromosome segregation during neurogenesis

Atkins

et al. 2020

eLife

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Mutations of SMC5/6 components cause developmental defects, including primary microcephaly. To model neurodevelopmental defects, we engineered a mouse wherein Smc5 is conditionally knocked out (cKO) in the developing neocortex. Smc5 cKO mice exhibited neurodevelopmental defects due to neural progenitor cell (NPC) apoptosis, which led to reduction in cortical layer neurons. Smc5 cKO NPCs formed DNA bridges during mitosis and underwent chromosome missegregation. SMC5/6 depletion triggers a CHEK2-p53 DNA damage response, as concomitant deletion of the Trp53 tumor suppressor or Chek2 DNA damage checkpoint kinase rescued Smc5 cKO neurodevelopmental defects. Further assessment using Smc5 cKO and auxin-inducible degron systems demonstrated that absence of SMC5/6 leads to DNA replication stress at late-replicating regions such as pericentromeric heterochromatin regions. In summary, SMC5/6 is important for completion of DNA replication prior to entering mitosis, which ensures accurate chromosome segregation. Thus, SMC5/6 functions are critical in highly proliferative stem cells during organism development.

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Section: Mesc Culture and Analysismentioning

confidence: 99%

SMC5/6 is required for replication fork stability and faithful chromosome segregation during neurogenesis

Atkins

et al. 2020

eLife

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“…We feel that this fact is either omitted or not stressed enough in most published protocols (Natsume et al, 2016;Lambrus et al, 2018;Yesbolatova et al, 2019;Sathyan et al, 2020). Our observations also warn against removing the selection cassette after AFB integration in safeharbor loci (Pryzhkova et al, 2020). In the future, exclusion of the AFB expression step could help to improve the AID experience (Yamanaka et al, 2020), but the method is still under development.…”

Section: Discussionmentioning

confidence: 94%

“…The AID system has been successfully applied in many organisms ranging from yeast to transgenic mice ( Yesbolatova et al, 2020 ). Adaptation of the AID system in popular cell cultures, such as HeLa, HCT116, mouse and human stem cells, and neurons ( Natsume et al, 2016 ; Nora et al, 2017 ; Nakano et al, 2019 ; Aksenova et al, 2020 ; Pryzhkova et al, 2020 ; Thiecke et al, 2020 ), was extremely fruitful, which allowed to study complex molecular pathways for virtually unlimited applications.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of the OsTIR1 and AtAFB2 AID Systems for Genome Architectural Protein Degradation in Mammalian Cells

Yunusova

Smirnov

Shnaider

et al. 2021

Front. Mol. Biosci.

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The auxin-inducible degron (AID) system is a promising tool for dynamic protein degradation. In mammalian cells, this approach has become indispensable to study fundamental molecular functions, such as replication, chromatin dynamics, or transcription, which are otherwise difficult to dissect. We present evaluation of the two prominent AID systems based on OsTIR1 and AtAFB2 auxin receptor F-box proteins (AFBs). We analyzed degradation dynamics of cohesin/condensin complex subunits in mouse embryonic stem cells (Rad21, Smc2, Ncaph, and Ncaph2) and human haploid HAP1 line (RAD21, SMC2). Double antibiotic selection helped achieve high homozygous AID tagging of an endogenous gene for all genes using CRISPR/Cas9. We found that the main challenge for successful protein degradation is obtaining cell clones with high and stable AFB expression levels due to the mosaic expression of AFBs. AFB expression from a transgene tends to decline with passages in the absence of constant antibiotic selection, preventing epigenetic silencing of a transgene, even at the AAVS1 safe-harbor locus. Comparing two AFBs, we found that the OsTIR1 system showed weak dynamics of protein degradation. At the same time, the AtAFB2 approach was very efficient even in random integration of AFB-expressed transgenes. Other factors such as degradation dynamics and low basal depletion were also in favor of the AtAFB2 system.

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“…In theory, integration of the AFBs in the safe-harbor locus should provide sufficient expression levels. This approach is recommended in majority of protocols where many suitable loci (H11, TIGRE, AAVS1, ROSA26) were extensively characterized [4,7,19]. The downsides of the safe-harbor targeting approach are extra experimental manipulations, restricted expression levels, and susceptibility to silencing, just as in randomly integrated transgene constructs (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…We feel that this fact is either omitted or not stressed enough in most published protocols [3,9,11,12]. Our observations also warn against removing selection cassette after AFB integration in safe-harbor loci [7]. In future, exclusion of the AFB expression step could help to improve the AID experience [20], but the method is still under development.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of the OsTIR1 and AtAFB2 AID systems for chromatin protein degradation in mammalian cells

Yunusova

Smirnov

Shnaider

et al. 2021

Preprint

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Auxin-inducible degron (AID) system is a promising tool for dynamic protein degradation. In mammalian cells, this approach has become indispensable to study fundamental molecular functions, such as replication, chromatin dynamics or transcription, that are otherwise difficult to dissect. We present evaluation of the two prominent AID systems based on OsTIR1 and AtAFB2 auxin receptor F-box proteins (AFBs). We analyzed degradation dynamics of cohesin/condensin complexes subunits in mouse embryonic stem cells (mRad21, mSMC2, mCapH, mCapH2) and human haploid HAP1 line (hRad21, hSMC2). Double antibiotic selection helped to achieve high homozygous AID targeting efficiency for all genes, ranging from 11 to 77%. We found that the main challenge for successful protein degradation is obtaining cell clones with high and stable AFB expression levels due to mosaic expression of AFBs, which also tends to decline with passages in the absence of constant puromycin selection, even at the AAVS1 safe-harbor locus. Comparing two AFBs, we found that OsTIR1 system showed weak dynamics of protein degradation. At the same time, AtAFB2 approach was very efficient even in random integration. Other factors such as degradation dynamics and low basal depletion were also in favor of AtAFB2 system. Our main conclusion is that repeated addition of puromycin to culture medium prevents AtAFB2 silencing and restores auxin sensitivity, facilitating robust protein degradation. We hope that our report will be useful for researchers that plan to establish AID method in their lab.

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Adaptation of the AID system for stem cell and transgenic mouse research

Cited by 9 publications

References 79 publications

SMC5/6 is required for replication fork stability and faithful chromosome segregation during neurogenesis

SMC5/6 is required for replication fork stability and faithful chromosome segregation during neurogenesis

Evaluation of the OsTIR1 and AtAFB2 AID Systems for Genome Architectural Protein Degradation in Mammalian Cells

Evaluation of the OsTIR1 and AtAFB2 AID systems for chromatin protein degradation in mammalian cells

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