Uncovering the fine print of the CreER^T2<i>-LoxP</i> system while generating a conditional knockout mouse model of <i>Ssrp1</i> gene

<div><p><b>FA</b>cilitates <b>C</b>hromatin <b>T</b>ranscription (<b>FACT</b>) is a complex of SSRP1 and SPT16 that is involved in chromatin remodeling during transcription, replication, and DNA repair. FACT has been mostly studied in cell-free or single cell model systems because general FACT knockout (KO) is embryonically lethal (E3.5). FACT levels are limited to the early stages of development and stem cell niches of adult tissues. FACT is upregulated in poorly differentiated aggressive tumors. Importantly, FACT inhibition (RNAi) is lethal for tumors but not normal cells, making FACT a lucrative target for anticancer therapy. To develop a better understanding of FACT function in the context of the mammalian organism under normal physiological conditions and in disease, we aimed to generate a conditional FACT KO mouse model. Because SPT16 stability is dependent on the SSRP1-SPT16 association and the presence of <i>SSRP1</i> mRNA, we targeted the <i>Ssrp1</i> gene using a CreER^T2- LoxP approach to generate the FACT KO model. Here, we highlight the limitations of the CreER^T2-LoxP (Rosa26) system that we encountered during the generation of this model. <i>In vitro</i> studies showed an inefficient excision rate of ectopically expressed CreER^T2 (retroviral CreER^T2) in fibroblasts with homozygous floxed <i>Ssrp1</i>. <i>In vitro</i> and <i>in vivo</i> studies showed that the excision efficiency could only be increased with germline expression of two alleles of Rosa26CreER^T2. The expression of one germline Rosa26CreER^T2 allele led to the incomplete excision of <i>Ssrp1</i>. The limited efficiency of the CreER^T2-LoxP system may be sufficient for studies involving the deletion of genes that interfere with cell growth or viability due to the positive selection of the phenotype. However, it may not be sufficient for studies that involve the deletion of genes supporting growth, or those crucial for development. Although CreER^T2-LoxP is broadly used, it has limitations that have not been widely discussed. This paper aims to encourage such discussions.</p></div

Excision efficiency of two alleles of germline <i>CreER</i>^<i>T2</i> in vivo.

<p>A) PCR of genomic DNA to determine <i>Ssrp1</i> excision in <i>Ssrp1</i>^<i>fl/fl</i> <i>CreER</i>^<i>T2</i>+/- and <i>Ssrp1</i>^<i>fl/fl</i> <i>CreER</i>^<i>T2</i>+/+ mice treated with 1 mg tamoxifen or <i>Ssrp1</i>^<i>fl/fl</i><i>CreER</i>^<i>T2</i>+/- mice treated with vehicle control i.p. for 5 days. T = tamoxifen-treated mice and C = vehicle-treated control mice; T2 = <i>Ssrp1</i>^<i>fl/fl</i> <i>CreER</i>^<i>T2</i>+/ mouse treated twice with tamoxifen. For T2, the image shows the PCR results following the second round of treatment.^-B) qPCR to determine the relative expression of the excised gene in <i>Ssrp1</i>^<i>fl/fl</i> <i>CreER</i>^<i>T2</i>+/- and <i>Ssrp1</i>^<i>fl/fl</i> <i>CreER</i>^<i>T2</i>+/+ mice treated with 1 mg tamoxifen i.p. for 5 days. p-value = 0.0022.</p

Excision efficiency of ectopically expressed CreER^T2 (MSCV CreER^T2).

<p>A) Methylene Blue staining of immortalized and transformed <i>Ssrp1</i>^<i>fl/fl</i> fibroblasts and immunoblot determining the levels of SSRP1 and SPT16 in primary, immortalized and transformed cells. B) Immunoblot to determine time-dependent changes in SSRP1 protein levels in immortalized Ssrp1^fl/fl MSCV CreER^T2 upon treatment with 2 μM 4-OHT. C) SSRP1 Immunofluorescence in immortalized and transformed <i>Ssrp1</i> ^<i>fl/fl</i> +/- MSCV CreER^T2 before or after treatment with 2 μM 4-OHT for 72 or 96 h. D) Quantification of SSRP1 immunofluorescence staining to calculate the Corrected Total Cell Fluorescence (CTCF). E) PCR of the genomic DNA to determine the excision of <i>Ssrp1</i> after 4-OHT treatment of immortalized and transformed <i>Ssrp1</i>^<i>fl/fl</i> fibroblasts with or without transduction with MSCV CreER^T2.</p

Liquid Deposition Patterning of Conducting Polymer Ink onto Hard and Soft Flexible Substrates via Dip-Pen Nanolithography

Ink formulations and protocols that enable the deposition and patterning of a conducting polymer (PEDOT:PSS) in the nanodomain have been developed. Significantly, we demonstrated the ability to pattern onto soft substrates such as silicone gum and polyethylene terephthalate (PET), which are materials of interest for low cost, flexible electronics. The deposition process and dimensions of the polymer patterns are found to be critically dependent on a number of parameters, including the pen design, ink properties, time after inking the pen, dwell time of the pen on the surface, and the nature of material substrate. By assessing these different parameters, an improved understanding of the ability to control the dimensions of individual PEDOT:PSS structures down to 600 nm in width and 10–80 nm in height within patterned arrays was obtained. This applicability of DPN for simple and nonreactive liquid deposition patterning of conducting polymers can lead to the fabrication of organic nanoelectronics or biosensors and complement the efforts of existing printing techniques such as inkjet and extrusion printing by scaling down conductive components to submicrometer and nanoscale dimensions

Generation of conditional <i>Ssrp1</i> KO mice.

<p>A) Schematic representation of the wild-type <i>Ssrp1</i> allele (<i>Ssrp1</i>⁺) and the targeting vector with 5’ and 3’ homology arms (pink dashed lines). The black dashed lines indicate intron regions in <i>Ssrp1</i>⁺ where LoxP sites, FRT sites, and the synthetic cassette are inserted. B) Schematic representation of the mutant allele (<i>Ssrp1</i> ^{<i>Neo LacZ fl</i>}) after homologous recombination, representation of mutant allele without the synthetic cassette (<i>Ssrp1</i>^<i>fl</i>) after Flp-FRT recombination and representation of <i>Ssrp1</i>^<i>Δ</i> allele with deletion of the critical exons after CreER^T2-LoxP recombination. C) Southern blot hybridization of DNA from ESC cells to determine clones with correct (red) and incorrect (black) recombination of 3’ and 5’ arm. Wild-type DNA used as control for 5’ probe is C. D) PCR of genomic DNA from F1 progeny of wild-type (<i>Ssrp1</i>^<i>+/+</i>) crossed with chimera (<i>Ssrp1</i> ^{<i>Neo LacZ fl /</i>+}). Two bands are present in heterozygous pups (<i>Ssrp1</i> ^{<i>Neo LacZ fl</i> /+}) (red). Numbers indicate individual pup IDs in one litter. E) β-Galactosidase assay to determine LacZ activity (Blue-Green stain for positive activity) on SSRP1 positive (colon, pancreas and testis) and negative (lung) tissues isolated from <i>Ssrp1</i>^+/+ and <i>Ssrp1</i> ^{<i>Neo LacZ fl</i> /+} mice.</p

Mosaic excision of Ssrp1 in a clone generated by limiting dilution.

<p>An example of a clone developing from a single transformed <i>Ssrp1</i>^<i>fl/fl</i> MSCV CreER^T2 cell. SSRP1 immunofluorescence (green) in the presence and absence of treatment with 2 μM 4-OHT for 120 h. DNA was counterstained with Hoechst.</p

Excision efficiency of <i>Ssrp1</i> ^<i>fl/fl</i> by two alleles of germline-expressed <i>CreER</i>^<i>T2</i>.

<p>A) Immunoblot to determine SSRP1 and SPT16 protein expression in primary, immortalized, and transformed <i>Ssrp1</i>^<i>fl/fl</i> <i>CreER</i>^<i>T2</i>+/+ and <i>Ssrp1</i> ^<i>fl/+</i> <i>CreER</i>^<i>T2</i>+/+ fibroblasts with or without 4-OHT treatment (2 μM) for 120 h. B) PCR of genomic DNA to confirm excision of Ssrp1 in primary, immortalized, and transformed <i>Ssrp1</i>^<i>fl/fl</i> <i>CreER</i>^<i>T2</i>+/+ and <i>Ssrp1</i> ^<i>fl/+</i> <i>CreER</i>^<i>T2</i>+/+ fibroblasts with or without 4-OHT treatment (2 μM) for 120 h. C) qPCR to determine the relative expression of the excised gene in <i>Ssrp1</i>^<i>fl/fl</i> MSCV CreER^T2, <i>Ssrp1</i>^<i>fl/fl</i> <i>CreER</i>^<i>T2</i>+/- and <i>Ssrp1</i>^<i>fl/fl</i> <i>CreER</i>^<i>T2</i>+/+ cells when treated with 2 μM 4-OHT for 120 h.</p

Effect of TSA on <i>Ssrp1</i>^<i>fl</i> excision by ectopic and germline CreER^T2.

<p>A) Immunoblot of SSRP1 protein expression in transformed <i>Ssrp1</i>^<i>fl/fl</i> MSCV CreER^T2 cells with or without 4-OHT and TSA treatment. B) PCR of genomic DNA of transformed <i>Ssrp1</i>^<i>fl/fl</i> MSCV CreER^T2 cells with or without 4-OHT and TSA treatment. C) PCR of genomic DNA of immortalized <i>Ssrp1</i>^<i>fl/fl</i> <i>CreER</i>^<i>T2</i>+/- with or without 2 μM 4-OHT treatment for 120 h. D) Immunoblot of SSRP1 protein expression in immortalized <i>Ssrp1</i>^<i>fl/fl</i> <i>CreER</i>^<i>T2</i>+/- cells with or without 4-OHT and TSA treatment. E) PCR of genomic DNA of immortalized <i>Ssrp1</i>^<i>fl/fl</i> <i>CreER</i>^<i>T2</i>+/- cells with or without 4-OHT and TSA treatment.</p

Vapor Phase Polymerization of EDOT from Submicrometer Scale Oxidant Patterned by Dip-Pen Nanolithography

Some of the most exciting recent advances in conducting polymer synthesis have centered around the method of vapor phase polymerization (VPP) of thin films. However, it is not known whether the VPP process can proceed using significantly reduced volumes of oxidant and therefore be implemented as part of nanolithography approach. Here, we present a strategy for submicrometer scale patterning of the conducting polymer poly­(3,4-ethylenedioxythiophene) (PEDOT) via in situ VPP. Attolitre (10^–18 L) volumes of oxidant “ink” are controllably deposited using dip-pen nanolithography (DPN). DPN patterning of the oxidant ink is facilitated by the incorporation of an amphiphilic block copolymer thickener, an additive that also assists with stabilization of the oxidant. When exposed to EDOT monomer in a VPP chamber, each deposited feature localizes the synthesis of conducting PEDOT structures of several micrometers down to 250 nm in width. PEDOT patterns are characterized by atomic force microscopy (AFM), conductive AFM, two probe electrical measurement, and micro-Raman spectroscopy, evidencing in situ vapor phase synthesis of conducting polymer at a scale (picogram) which is much smaller than that previously reported. Although the process of VPP on this scale was achieved, we highlight some of the challenges that need to be overcome to make this approach feasible in an applied setting

Pairing is not limited to monoallelically expressed regions.

<p><b>A</b>) Extent of paired region on chromosome 7 in ES cells. Each dot represents the mean of three to four samples taken from different passages with the pairing frequency determined for each sample in four technical replicates of 300 nuclei. Whiskers represent standard deviation. Probes from the imprinted region on distal chromosome 7 are shown as open circles, probes from non-imprinted regions are shown as filled circles. Background shading indicates high pairing frequency (dark grey, above 3.5%), medium (light grey, 2.5–3.5%) and low pairing frequency (yellow, below 2.5%). The myc probe (chromosome 15) is displayed for comparison. Frequent pairing is observed for the distal end of chromosome 7 but not limited to the imprinted region. <b>B</b>) Pairing in imprinting deficient mutants. Each dot represents one biological sample with the pairing frequency determined in four technical replicates of 300 nuclei. The mean is represented by a line. Differences were assessed by unpaired t-test, ns: not significant. Wt: wildtype, KvDMR del: paternal deletion of KvDMR abolishing regional silencing and removing functionally important CTCF binding sites, RNA trunc: paternal truncation of the non-coding RNA Kcnq1ot1 responsible for monoallelic silencing, par ES: parthenogenetic ES cells harbouring two maternal genomes, andr ES: androgenetic ES cells harbouring two paternal genomes. Neither the monoallelic expression state nor the presence of a biparental genome is a prerequisite for frequent pairing in the region. <b>C</b>) Pairing of other imprinted and telomeric regions in ES cells. For a description of data points and background see A. Differences were assessed by unpaired t-test, ns: not significant, *: p<0.05. Imprinted regions are represented by open circles, regions close to the telomere by filled diamonds. Probes covering genes in various imprinted regions do not display high pairing frequency in contrast to probes located near telomeres.</p