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

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.

Download Full-text

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

10.1101/2021.03.15.435352 ◽

2021 ◽

Author(s):

Anastasia Yunusova ◽

Alexander Smirnov ◽

Tatiana Shnaider ◽

Svetlana Afonnikova ◽

Nariman Battulin

Keyword(s):

Protein Degradation ◽

Mammalian Cells ◽

Embryonic Stem ◽

Auxin Receptor ◽

Chromatin Dynamics ◽

Promising Tool ◽

Main Challenge ◽

Cell Clones ◽

Mosaic Expression ◽

Puromycin Selection

ABSTRACTAuxin-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.

Download Full-text

Gene Targeting

10.1093/oso/9780199637928.001.0001 ◽

1999 ◽

Cited By ~ 2

Keyword(s):

Homologous Recombination ◽

Gene Targeting ◽

Mammalian Cells ◽

Es Cells ◽

Practical Approach ◽

Simple Method ◽

Mouse Es Cells ◽

Cell Clones ◽

Previous Edition ◽

Pros And Cons

Since the publication of the first edition of Gene Targeting: A Practical Approach in 1993 there have been many advances in gene targeting and this new edition has been thoroughly updated and rewritten to include all the major new techniques. It provides not only tried-and-tested practical protocols but detailed guidance on their use and applications. As with the previous edition Gene Targeting: A Practical Approach 2e concentrates on gene targeting in mouse ES cells, but the techniques described can be easily adapted to applications in tissue culture including those for human cells. The first chapter covers the design of gene targeting vectors for mammalian cells and describes how to distinguish random integrations from homologous recombination. It is followed by a chapter on extending conventional gene targeting manipulations by using site-specific recombination using the Cre-loxP and Flp-FRT systems to produce 'clean' germline mutations and conditionally (in)activating genes. Chapter 3 describes methods for introducing DNA into ES cells for homologous recombination, selection and screening procedures for identifying and recovering targeted cell clones, and a simple method for establishing new ES cell lines. Chapter 4 discusses the pros and cons or aggregation versus blastocyst injection to create chimeras, focusing on the technical aspects of generating aggregation chimeras and then describes some of the uses of chimeras. The next topic covered is gene trap strategies; the structure, components, design, and modification of GT vectors, the various types of GT screens, and the molecular analysis of GT integrations. The final chapter explains the use of classical genetics in gene targeting and phenotype interpretation to create mutations and elucidate gene functions. Gene Targeting: A Practical Approach 2e will therefore be of great value to all researchers studying gene function.

Download Full-text

Orthogonal control of mean and variability of endogenous genes in a human cell line

Nature Communications ◽

10.1038/s41467-020-20467-8 ◽

2021 ◽

Vol 12 (1) ◽

Cited By ~ 1

Author(s):

Alain R. Bonny ◽

João Pedro Fonseca ◽

Jesslyn E. Park ◽

Hana El-Samad

Keyword(s):

Mammalian Cells ◽

Human Cell Line ◽

Transcriptional Level ◽

Clear Understanding ◽

Gene Expression Noise ◽

Endogenous Gene ◽

Basal Expression ◽

Stochastic Fluctuations ◽

Human Genes ◽

Synthetic Circuit

AbstractStochastic fluctuations at the transcriptional level contribute to isogenic cell-to-cell heterogeneity in mammalian cell populations. However, we still have no clear understanding of the repercussions of this heterogeneity, given the lack of tools to independently control mean expression and variability of a gene. Here, we engineer a synthetic circuit to modulate mean expression and heterogeneity of transgenes and endogenous human genes. The circuit, a Tunable Noise Rheostat (TuNR), consists of a transcriptional cascade of two inducible transcriptional activators, where the output mean and variance can be modulated by two orthogonal small molecule inputs. In this fashion, different combinations of the inputs can achieve the same mean but with different population variability. With TuNR, we achieve low basal expression, over 1000-fold expression of a transgene product, and up to 7-fold induction of the endogenous gene NGFR. Importantly, for the same mean expression level, we are able to establish varying degrees of heterogeneity in expression within an isogenic population, thereby decoupling gene expression noise from its mean. TuNR is therefore a modular tool that can be used in mammalian cells to enable direct interrogation of the implications of cell-to-cell variability.

Download Full-text

Simple and versatile imaging of genomic loci in live mammalian cells and early pre-implantation embryos using CAS-LiveFISH

Scientific Reports ◽

10.1038/s41598-021-91787-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Yongtao Geng ◽

Alexandros Pertsinidis

Keyword(s):

Mammalian Cells ◽

Target Gene ◽

Nuclear Architecture ◽

Live Cells ◽

Transcription Activator ◽

Distal Enhancer ◽

Chromatin Dynamics ◽

Guide Rnas ◽

Early Mouse

AbstractVisualizing the 4D genome in live cells is essential for understanding its regulation. Programmable DNA-binding probes, such as fluorescent clustered regularly interspaced short palindromic repeats (CRISPR) and transcription activator-like effector (TALE) proteins have recently emerged as powerful tools for imaging specific genomic loci in live cells. However, many such systems rely on genetically-encoded components, often requiring multiple constructs that each must be separately optimized, thus limiting their use. Here we develop efficient and versatile systems, based on in vitro transcribed single-guide-RNAs (sgRNAs) and fluorescently-tagged recombinant, catalytically-inactivated Cas9 (dCas9) proteins. Controlled cell delivery of pre-assembled dCas9-sgRNA ribonucleoprotein (RNP) complexes enables robust genomic imaging in live cells and in early mouse embryos. We further demonstrate multiplex tagging of up to 3 genes, tracking detailed movements of chromatin segments and imaging spatial relationships between a distal enhancer and a target gene, with nanometer resolution in live cells. This simple and effective approach should facilitate visualizing chromatin dynamics and nuclear architecture in various living systems.

Download Full-text

Target frequency and integration pattern for insertion and replacement vectors in embryonic stem cells

Molecular and Cellular Biology ◽

10.1128/mcb.11.9.4509-4517.1991 ◽

1991 ◽

Vol 11 (9) ◽

pp. 4509-4517

Author(s):

P Hasty ◽

J Rivera-Pérez ◽

C Chang ◽

A Bradley

Keyword(s):

Homologous Recombination ◽

Gene Targeting ◽

Mammalian Cells ◽

Germ Line ◽

Es Cells ◽

Embryonic Stem ◽

Homologous Sequence ◽

Reciprocal Recombination ◽

Replacement Vector ◽

Insertion Vector

Gene targeting has been used to direct mutations into specific chromosomal loci in murine embryonic stem (ES) cells. The altered locus can be studied in vivo with chimeras and, if the mutated cells contribute to the germ line, in their offspring. Although homologous recombination is the basis for the widely used gene targeting techniques, to date, the mechanism of homologous recombination between a vector and the chromosomal target in mammalian cells is essentially unknown. Here we look at the nature of gene targeting in ES cells by comparing an insertion vector with replacement vectors that target hprt. We found that the insertion vector targeted up to ninefold more frequently than a replacement vector with the same length of homologous sequence. We also observed that the majority of clones targeted with replacement vectors did not recombine as predicted. Analysis of the recombinant structures showed that the external heterologous sequences were often incorporated into the target locus. This observation can be explained by either single reciprocal recombination (vector insertion) of a recircularized vector or double reciprocal recombination/gene conversion (gene replacement) of a vector concatemer. Thus, single reciprocal recombination of an insertion vector occurs 92-fold more frequently than double reciprocal recombination of a replacement vector with crossover junctions on both the long and short arms.

Download Full-text

Non-invasive somatotransgenic bioimaging in living animals

F1000Research ◽

10.12688/f1000research.25274.1 ◽

2020 ◽

Vol 9 ◽

pp. 1216 ◽

Cited By ~ 1

Author(s):

Juliette M. Delhove ◽

Rajvinder Karda ◽

Lorna M. FitzPatrick ◽

Suzanne M.K. Buckley ◽

Simon N. Waddington ◽

...

Keyword(s):

Gene Expression ◽

Reporter Gene ◽

Viral Vectors ◽

Es Cells ◽

Embryonic Stem ◽

Whole Body ◽

Luciferase Reporter ◽

Gene Promoters ◽

Luciferase Reporter Gene ◽

Endogenous Gene

Bioluminescence imaging enables noninvasive quantification of luciferase reporter gene expression in transgenic tissues of living rodents. Luciferase transgene expression can be regulated by endogenous gene promoters after targeted knock-in of the reporter gene, usually within the first intron of the gene. Even using CRISPR/Cas9 mediated genome editing this can be a time consuming and costly process. The generation of germline transgenic (GLT) rodents by targeted genomic integration of a gene expression cassette in embryonic stem (ES) cells is commonplace but results in the wastage of large numbers of animals during colony generation, back-crossing and maintenance. Using a synthetic/truncated promoter-driven luciferase gene to study promoter activity in a given tissue or organ of a GLT also often results in unwanted background luciferase activity during whole-body bioluminescent imaging as every cell contains the reporter. We have developed somatotransgenic bioimaging; a method to generate tissue-restricted transcription factor activated luciferase reporter (TFAR) cassettes in rodents that substantially reduces the number of animals required for experimentation. Bespoke designed TFARs are delivered to newborn pups using viral vectors targeted to specific organs by tissue-tropic pseudotypes. Retention and proliferation of TFARs is facilitated by stem/progenitor cell transduction and immune tolerance to luciferase due to the naïve neonatal immune system. We have successfully applied both lentiviral and adeno-associated virus (AAV) vectors in longitudinal rodent studies, targeting TFARs to the liver and brain during normal development and in well-established disease models. Development of somatotransgenic animals has broad applicability to non-invasively determine mechanistic insights into homeostatic and disease states and assess toxicology and efficacy testing. Somatotransgenic bioimaging technology is superior to current whole-body, light-emitting transgenic models as it reduces the numbers of animals used by generating only the required number of animals. It is also a refinement over current technologies given the ability to use conscious, unrestrained animals.

Download Full-text

Characterization of constitutive HSF2 DNA-binding activity in mouse embryonal carcinoma cells

Molecular and Cellular Biology ◽

10.1128/mcb.14.8.5309-5317.1994 ◽

1994 ◽

Vol 14 (8) ◽

pp. 5309-5317

Author(s):

S P Murphy ◽

J J Gorzowski ◽

K D Sarge ◽

B Phillips

Keyword(s):

Transcription Factors ◽

Heat Shock ◽

Dna Binding ◽

Mammalian Cells ◽

Embryonal Carcinoma ◽

Embryonic Stem ◽

Binding Activity ◽

Hsp70 Promoter ◽

Ec Cells

Two distinct murine heat shock transcription factors, HSF1 and HSF2, have been identified. HSF1 mediates the transcriptional activation of heat shock genes in response to environmental stress, while the function of HSF2 is not understood. Both factors can bind to heat shock elements (HSEs) but are maintained in a non-DNA-binding state under normal growth conditions. Mouse embryonal carcinoma (EC) cells are the only mammalian cells known to exhibit HSE-binding activity, as determined by gel shift assays, even when maintained at normal physiological temperatures. We demonstrate here that the constitutive HSE-binding activity present in F9 and PCC4.aza.R1 EC cells, as well as a similar activity found to be present in mouse embryonic stem cells, is composed predominantly of HSF2. HSF2 in F9 EC cells is trimerized and is present at higher levels than in a variety of nonembryonal cell lines, suggesting a correlation of these properties with constitutive HSE-binding activity. Surprisingly, transcription run-on assays suggest that HSF2 in unstressed EC cells does not stimulate transcription of two putative target genes, hsp70 and hsp86. Genomic footprinting analysis indicates that HSF2 is not bound in vivo to the HSE of the hsp70 promoter in unstressed F9 EC cells, although HSF2 is present in the nucleus and the promoter is accessible to other transcription factors and to HSF1 following heat shock. Thus trimerization and nuclear localization of HSF2 do not appear to be sufficient for in vivo binding of HSF2 to the HSE of the hsp70 promoter in unstressed F9 EC cells.

Download Full-text

The T gene is necessary for normal mesodermal morphogenetic cell movements during gastrulation

Development ◽

10.1242/dev.121.3.877 ◽

1995 ◽

Vol 121 (3) ◽

pp. 877-886 ◽

Cited By ~ 27

Author(s):

V. Wilson ◽

L. Manson ◽

W.C. Skarnes ◽

R.S. Beddington

Keyword(s):

T Cells ◽

Neural Tube ◽

Es Cells ◽

Embryonic Stem ◽

Primitive Streak ◽

Distal Portion ◽

Adhesion Properties ◽

Cell Clones ◽

Posterior Neuropore ◽

Pass Through

The T (Brachyury) deletion in mouse is responsible for defective primitive streak and notochord morphogenesis, leading to a failure of the axis to elongate properly posterior to the forelimb bud. T/T embryonic stem (ES) cells colonise wild-type embryos, but in chimeras at 10.5 days post coitum (dpc) onwards they are found predominantly in the distal tail, while trunk paraxial and lateral mesoderm are deficient in T/T cells (Wilson, V., Rashbass, P. and Beddington, R. S. P. (1992) Development 117, 1321–1331). To determine the origin of this abnormal tissue distribution, we have isolated T/T and control T/+ ES cell clones which express lacZ constitutively using a gene trap strategy. Visualisation of T/T cell distribution in chimeric embryos throughout gastrulation up to 10.5 dpc shows that a progressive buildup of T/T cells in the primitive streak during gastrulation leads to their incorporation into the tailbud. These observations make it likely that one role of the T gene product is to act during gastrulation to alter cell surface (probably adhesion) properties as cells pass through the primitive streak. As the chimeric tail elongates at 10.5 dpc, abnormal morphology in the most distal portion becomes apparent. Comparison of T expression in the developing tailbud with the sites of accumulation of T/T cells in chimeras shows that T/T cells collect in sites where T would normally be expressed. T expression becomes internalised in the tailbud following posterior neuropore closure while, in abnormal chimeric tails, T/T cells remain on the surface of the distal tail. We conclude that prevention of posterior neuropore closure by the wedge of T/T cells remaining in the primitive streak after gastrulation is one source of the abnormal tail phenotypes observed. Accumulation of T/T cells in the node and anterior streak during gastrulation results in the preferential incorporation of T/T cells into the ventral portion of the neural tube and axial mesoderm. The latter forms compact blocks which are often fused with the ventral neural tube, reminiscent of the notochordal defects seen in intact mutants. Such fusions may be attributed to cell-autonomous changes in cell adhesion, possibly related to those observed at earlier stages in the primitive streak.

Download Full-text

Effect of NANOG overexpression on porcine embryonic development and pluripotent embryonic stem cell formation in vitro

Zygote ◽

10.1017/s0967199421000678 ◽

2021 ◽

pp. 1-6

Author(s):

Gerelchimeg Bou ◽

Shimeng Guo ◽

Jia Guo ◽

Zhuang Chai ◽

Jianchao Zhao ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Embryonic Stem Cells ◽

Embryonic Stem Cell ◽

Embryonic Stem Cell Line ◽

Embryonic Stem ◽

Cell Clones ◽

Morula Stage ◽

Pluripotent Embryonic Stem Cell

Summary The efficiency of establishing pig pluripotent embryonic stem cell clones from blastocysts is still low. The transcription factor Nanog plays an important role in maintaining the pluripotency of mouse and human embryonic stem cells. Adequate activation of Nanog has been reported to increase the efficiency of establishing mouse embryonic stem cells from 3.5 day embryos. In mouse, Nanog starts to be strongly expressed as early as the morula stage, whereas in porcine NANOG starts to be strongly expressed by the late blastocyst stage. Therefore, here we investigated both the effect of expressing NANOG on porcine embryos early from the morula stage and the efficiency of porcine pluripotent embryonic stem cell clone formation. Compared with intact porcine embryos, NANOG overexpression induced a lower blastocyst rate, and did not show any advantages for embryo development and pluripotent embryonic stem cell line formation. These results indicated that, although NANOG is important pluripotent factor, NANOG overexpression is unnecessary for the initial formation of porcine pluripotent embryonic stem cell clones in vitro.

Download Full-text