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Cite This: ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

Enhanced TAT-Cre Protein Transduction for Efficient Gene Recombination in T cells Federica Sgolastra,†,∥ Christina Arieta Kuksin,‡,∥ Gabriela Gonzalez-Perez,‡,∥ Lisa M. Minter,*,‡,§ and Gregory N. Tew*,†,‡,§ †

Department of Polymer Science & Engineering; ‡Department of Veterinary & Animal Science; §Program in Molecular & Cellular Biology, University of Massachusetts, Amherst, Massachusetts 01003, United States

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ABSTRACT: Genetic manipulation has increased our understanding of gene function and led to the discovery of new therapeutic targets. Cre/LoxP DNA recombination is widely used for genetic studies in mammalian cells. The direct delivery of Cre recombinase fused to protein transduction domains (PTDs), such as TAT, has been described as a valid alternative to the conditional, site-specific Cre expression in transgenic mice. However, efficiently conveying proteins into live cells, especially primary T cells, remains a major challenge. In this study, we show that one of our recently developed PTDs synthetic mimic greatly enhances the cellular uptake of the TAT-Cre fusion protein, enabling significantly smaller amounts of the protein to be used. We used this technique in primary mouse T cells to successfully delete, ex vivo, two essential genes involved in regulating T cell activation, Notch1 and Rbpjκ. Ex vivo gene deletion resulted in substantial protein reduction, comparable to that obtained in vivo when Cre-expressing Notch1-floxed (MxCre±Notch1f l/fl) mice were treated with polyinosinic-polycytidylic acid (polyl/ C), but in considerably less time, and without altering normal cell physiology. These results highlight several key advantages that include the ability to use less expensive protein (TAT-Cre), a major reduction in total experimental time and labor, and fewer side effects on the treated cells. This method should offer new opportunities for immunological studies, especially in the context of identifying novel therapeutic targets. KEYWORDS: Cre/LoxP, gene recombination, protein delivery, protein transduction domain mimics, TAT-Cre, primary T cells

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organisms3−6 and for a large number of biological applications, such as conditional transgenesis, selectable marker removal, gene knockout, and chromosome engineering.7 Within the context of immunology, Cre/LoxP DNA recombination (Scheme 1) has been used to investigate the contribution of specific genes in the development,8 maturation, activation,9 and differentiation10 of T cells, as well as to analyze the impact of cell signaling anomalies on normal mammalian development and function.11 Because T cells play a central role in the immune response to infection and in many autoimmune diseases, as well as their rising prominence in cancer treatment,12,13 the study of T cells and their signaling pathways is essential. For these studies, and also to avoid undesirable

INTRODUCTION Controlling gene expression is a powerful method to analyze mammalian gene function. It enables new understanding of cell signaling pathways and insight into the genetic basis of many diseases. As such, it holds great promise for the discovery of new therapeutic targets. However, manipulating the genome requires a multistep process, which is often laborious and timeconsuming. Various inducible systems have been engineered to express or delete a gene in a tissue- and time-specific manner.1 Among them, the Cre/LoxP strategy (Scheme 1) has gained popularity in cellular and molecular biology due to its simplicity and efficiency. The well-characterized Cre recombinase, derived from the Escherichia coli P1 bacteriophage, induces site-specific DNA recombination by recognizing and binding two 34-bp loxP sites.2 The recombination occurs between the loxP sites without the need of any additional cofactors. Cre expression has been used in a variety of © XXXX American Chemical Society

Received: May 23, 2018 Accepted: July 20, 2018

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DOI: 10.1021/acsabm.8b00153 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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Scheme 1. Schematic Illustration Highlighting the Noncovalent, Intracellular Delivery of Cre Recombinase by Protein Transduction Domain Mimicsa

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These mimics of cell penetrating peptide, or protein transduction domains, like HIV-TAT are more effective at intracellular delivery. In this study, Cre recombinase was delivered to demonstrate intracellular protein activity after delivery and to allow direct comparisons with the more traditional approach using promoters to induce Cre recombinase expression. The simplicity and effectiveness of this noncovalent approach is demonstrated in an important cell type, primary T cells.

especially important in T cells, given their primary role in immune response and the potential to interfere with the experimental design. Although engineered to be tissue- and/or time-specific, the control over this system is not always tight and a certain level of basal recombination can often be measured in untreated mice.19 Moreover, there is a risk of unintended recombination in response to endogenous IFN production in mice, especially in animals maintained in conventional facilities rather than under specific pathogenfree conditions. For some promoters, such as Mx1, induced Cre expression and consequent gene recombination can occur in more than one tissue and cell type at the same time, but with different efficiencies,19 driven by tissue-dependent IFN responsiveness or availability. All of these factors make it difficult to precisely and conveniently analyze the impact of gene deletion on individual cell types and in specific tissues, when inducing Cre expression in vivo. To circumvent these pitfalls, enzymatically active Cre protein has been directly delivered into cultured cells or mice by employing protein transduction domains (PTDs), also known as cell-penetrating peptides (CPPs).23,24 PTDs are short, cationic, cell-permeable peptides, which have been used to introduce large biologically active molecules into cells.25−27 Among them, the TAT peptide, derived from the HIV-1 transcriptional activator protein, has been extensively characterized for nonviral transduction of proteins and peptides.28,29 However, TAT must be covalently conjugated to the protein cargo, either chemically or genetically, in order to enable delivery.30 Despite early incorrect assumptions regarding the direct penetrability of TAT-fusion proteins across the cell membrane, recent evidence now points to lipid raft-dependent macropinocytosis as the dominant means of cellular uptake.31 Regardless of the exact mechanism, fusion proteins of Cre recombinase with TAT peptide (TAT-Cre) have been shown to enter cells and to induce site-specific recombination both in vitro using cell lines and ex vivo in embryonic stem cells and primary splenocytes.32,33 However, in those studies, TAT-Cre-

side effects that may result from constitutive Cre expression,14,15 a time- and tissue-specific DNA recombination is required. This is commonly achieved by generating transgenic mice carrying a Cre recombinase transgene under the control of a promoter that can be activated exclusively in immune tissues (such as thymus, spleen, or bone marrow) or at a particular maturation stage.16,17 In offspring derived from a cross between a Cre-expressing mouse and a mouse carrying a loxP-flanked (or “floxed”) target gene, Cre/LoxP recombination will take place only in those specific cells where Cre is expressed. Another strategy to achieve temporal control of gene deletion is to place Cre behind an inducible promoter. For example, the Mx1 promoter, which is activated by type 1 interferons (IFN), has been used to generate Mx-Cre transgenic mice. In these mice, Cre expression and subsequent DNA recombination is prompted in IFN-responsive tissues (such as liver and spleen) when IFN production is induced in vivo following repeated injections of polyl/C (polyinosinicpolycytidylic acid), or after direct injection of IFNα or IFNβ.18,19 These conditional knockout strategies are particularly useful to study genes that are essential for embryonic development, such as Notch1, in order to avoid the embryonic lethality associated with their lack of expression.20 Although inducible Cre expression has many benefits and is extensively used for in vivo gene deletion, there are significant drawbacks to this approach. Generating conditional Cre lines requires extensive mouse breeding, which is time-consuming, labor intensive, and expensive. Additionally, in order to efficiently activate an inducible promoter, such as Mx1, repeated IFN or polyl/C injections are needed. Subsequently, mice must be rested for up to 3 weeks to allow the inflammation induced by IFN production to subside,21 all of which adds to the experimental time and cost of using this method. Another point to consider is whether the substances used to induce Cre expression generate their own toxic side effects or alter the normal physiology of the mouse.22 This is B

DOI: 10.1021/acsabm.8b00153 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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“reporter” human T cell line, genetically modified to express the gene for the green fluorescent protein (GFP), floxed by two loxP sites, to demonstrate that combining our PTDM with a reduced amount of TAT-Cre significantly improves in vitro gene deletion compared to TAT-Cre alone. We further show this technology can be used ex vivo with primary mouse T cells to effectively delete floxed Notch1 and Rbpjκ, two genes important for T cell activation, differentiation, and survival,39 thus demonstrating the applicability of this method for immunological studies. Overall, our data indicate that the PTDM/TAT-Cre complex significantly improves Cre-mediated recombination by directly delivering active TAT-Cre into cells and, as such, represents a valid and advantageous alternative to the conventional, more laborious induction of Cre expression in vivo. One of the major benefits of this approach is a significant reduction in the experimental time required. In fact, high recombination efficiencies were obtained after only a few hours following treatment ex vivo with the PTDM/TAT-Cre system, compared to the three week time period required to delete these genes in vivo by treating mice with polyl/C. Additional benefits to using this approach include reduced time and costs associated with maintaining Mx-Cre transgenic mouse strains and breeding them to mice with floxed genes to generate inducible knockout strains. These PTDM/TAT-Cre complexes also required significantly less TAT-Cre, reducing its amount from micromolar to nanomolar concentrations. Because of the considerable advantages afforded by this new approach, we believe it will have broad applications in studies requiring ex vivo deletion of floxed genes, supporting novel target identification for therapeutic purposes.

promoted recombination was shown to be concentrationdependent and required micromolar concentrations to afford high recombination efficiencies (∼90%; see also Figure S1). We previously developed guanidinium-containing synthetic polymers designed to be PTD mimics (PTDMs).34,35 Recent optimization of their design for the use as noncovalent protein carriers has included the PTDM shown in Scheme 2.36,37 Scheme 2. Chemical Structure of the PTDM Used To Deliver TAT-Crea

The PTDM is a “gradient” or statistical copolymer synthesized using ring-opening metathesis polymerization.38

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According to this strategy, there is no need for the carrier and the cargo to be covalently fused together; rather, they form a supramolecular complex through noncovalent interactions (hydrophobic, H-bonding, and electrostatic), making this system more facile, versatile, and experimentally convenient. Moreover, when used to deliver the TAT-Cre fusion protein to a reporter cell line, these novel PTDMs were able to increase the efficacy of recombination and essentially abolish gene expression (∼95% reduction) using as little as 100 nM enzyme (Figure S2B).36 As a follow on to these encouraging preliminary results that showed enhanced gene recombination through PTDMmediated TAT-Cre delivery, we asked whether this approach could be used to easily and rapidly manipulate primary T cells (Scheme 1). Specifically, the results we obtained and report here represent a significant and important achievement, since nonviral manipulation of primary T cells remains an ill-defined and challenging area of investigation. Furthermore, we do not exclude the potential applicability of this method to other challenging cell types. In the present study, we utilized a

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RESULTS AND DISCUSSION PTDM Enhances TAT-Cre Transduction in a Reporter Cell Line in Vitro. The PTDM used here (Scheme 1) is based on a “gradient” or statistical copolymer design. This PTDM architecture was previously demonstrated to delivery EGFP similarly to its block copolymer constitutional macromolecular isomer.37,38 To optimize conditions for the PTDM-mediated delivery of TAT-Cre, we used a transgenic reporter human T cell line expressing the gene for GFP, floxed by two loxP sites. These cells constitutively express GFP but lose their

Figure 1. In vitro Cre-mediated recombination. GFP-floxed reporter T cells (2 × 105/mL) were exposed to 100 nM TAT-Cre alone (open symbol/bar) or combined with increasing amounts of PTDM (solid symbol/bars) for 2 h. Cell viability (represented on the right side y axis) was assessed by flow cytometry at the end of the 2 h treatment through AnnexinV and 7-AAD double-staining and compared to that of an untreated sample (A, dashed gray line). After 72 h, the percentage of GFP-expressing cells (represented on the left side y axis) was determined by flow cytometry (A, solid black line). Cell recovery was evaluated by comparing the number of viable cells after 72 h (determined by Trypan blue exclusion) to that of an untreated sample grown under the same conditions (B). Each point represents the mean ± SEM (standard error of mean) of three independent experiments. *(p < 0.05) when compared to TAT-Cre alone (white bar) as calculated by student’s t-test. C

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ACS Applied Bio Materials fluorescence after Cre-mediated recombination of the loxPGFP-loxP reporter gene. In this reporter system, exogenous, active Cre recombinase must enter the cells and reach the nucleus to delete the target gene. Therefore, the percentage of GFP-negative cells, as monitored by flow cytometry, represents a measure of cellular uptake efficiency. Although TAT-Cre fusion protein alone was able to enter cells and induce gene recombination in a concentration-dependent manner (Figure S1), consistent with previous studies, micromolar concentrations were necessary to obtain significant recombination. In fact, a minimum concentration of 1.5 μM (64.5 μg/mL) TATCre was required to achieve 90% reduction in GFP fluorescence, when it was delivered without the PTDM. In contrast, the amount of TAT-Cre necessary to induce this high degree of recombination was significantly reduced by following complexation with our synthetic PTDMs. Figure 1A shows a dose−response curve (solid black line) showing the degree of GFP recombination after cells were exposed for 2 h to as little as 100 nM TAT-Cre (4.3 μg/mL), with or without increasing amounts of PTDM. TAT-Cre alone induced recombination in less than 20% of the cell population (Figure 1A, black open circle), as expected. The addition of only 200 nM PTDM, corresponding to a 2:1 molar ratio with TAT-Cre, more than doubled the percentage of gene recombination (Figure 1A, black line). Increasing the PTDM/TAT-Cre molar ratio to 5:1 yielded approximately 90% recombination; however, further increasing the molar ratio to 10:1 led to only a limited additional improvement (∼95%, Figure 1A, black line). By comparison, delivering almost 20 times more TAT-Cre alone was necessary to achieve this same degree of recombination in the absence of PTDM (Figure S1). This increased efficiency for gene deletion highlights the advantage of PTDM-promoted TAT-Cre transduction in that significantly less protein is required to induce efficient recombination. Although the 5:1 PTDM/ TAT-Cre ratio caused some reduction in the number of live cells (∼20%) compared to PBS alone at 2 h post treatment, these cells fully recovered after 72 h (Figure 1A, dashed gray line; Figure 1B). Notably, only 2% of the cell death we observed was due to direct necrosis (Figure S3). Conversely, a higher molar ratio (10:1 PTDM/TAT-Cre) caused 30% reduction in viable cells 2 h after treatment, and these cells were unable to recover after 72 h (Figure 1B). The combination of highly efficient gene deletion and limited cell toxicity, together with the advantage of using less material, identified the 5:1 PTDM/TAT-Cre molar ratio as optimal and, therefore, was used for the remaining studies. Recombination-Signal Binding Protein-Jκ Reduction Following PTDM/TAT-Cre Treatment in Primary T Cells ex Vivo. We next investigated TAT-Cre-mediated recombination, ex vivo, in primary T cells isolated from the spleens of transgenic mice with or without the addition of the synthetic PTDM. It is well-known that primary T cells are an inherently difficult cell type to transduce.40 As a result, they represent an important challenge for this delivery system. We first used PTDM/TAT-Cre ex vivo with primary T cells isolated from Mx1-Cre−/−Rbpjκf l/fl mice. These transgenic mice are engineered to express homozygous Rbpjk alleles, flanked by LoxP sites, but they do not express Cre recombinase driven by the Mx1 promoter. Recombination-signal binding protein-Jκ (RBP-Jκ) is part of a transcriptional repressor complex, which is converted into a transcriptional activator when it associates with NOTCH signaling proteins in the nucleus, to mediate

what is known as canonical NOTCH signaling.41 In T cells, both canonical and noncanonical NOTCH signaling is vital for T cell development, activation, proliferation, and differentiation into various T helper subsets.10,42 The further delineation of canonical and noncanonical NOTCH signaling pathways will prove invaluable in developing therapies to target aberrant NOTCH signaling, which occurs in many human diseases. T cells were treated for 4 h with 250 nM TAT-Cre, alone or in combination with PTDM at a molar ratio of 5:1, in the presence of 10% FBS. The efficacy of Rbpjκ gene recombination was assessed at the protein level by Western blot 36 h after T cell stimulation. As shown in Figure 2, at the

Figure 2. RBP-Jκ expression after ex vivo Cre-mediated gene recombination. T cells were isolated from Mx1-Cre−/−Rbpjκfl/f l mice as described and plated in RDG medium with 10% FBS. TAT-Cre (250 nM), alone or in combination with 1.3 μM PTDM, was added. After 4 h of incubation, cells were washed and transferred to an antiCD3ε- and anti-CD28-coated plate for 36 h. RBP-Jκ expression was measured by Western blot (A) and normalized to actin levels (B). Each bar represents the mean + SEM of three independent experiments. *(p < 0.05); n.s. (p > 0.05) as calculated by student’s t-test.

concentration of TAT-Cre used, a significant increase in Rbpjk gene knock-down led to a greater than 50% reduction in protein expression (Figure 2B). No protein reduction was observed at 36 h with TAT-Cre alone, at the concentration used, in concordance with previous studies. Moreover, the protein reduction appeared to be site-specific, as expected, since the level of actin in the PTDM/TAT-Cre treated sample was equivalent to that of the blank (Figure 2A). As already demonstrated using the reporter cell line, the amount of PTDM used did not result in any significant reduction in cell viability compared to the control sample treated with PBS only (Figure S4). Ex Vivo vs in Vivo Cre-Mediated Notch1 Deletion. We next evaluated another primary target to ensure the approach described here was universal and that the measured protein knock-down was, indeed, derived from Cre-associated recombination at the loxP sites. T cells were isolated from mice engineered to express Notch1 flanked by two loxP sequences, but which lacked Mx1-Cre (Mx1-Cre−/−Notch1f l/fl). NOTCH1 belongs to a family of transmembrane receptors that are evolutionarily conserved and critically important to an array of biological functions. NOTCH signaling has been shown to play important roles in T cell-mediated autoimmune disease,21,39 and mutations of Notch are associated with higher incidence of many cancers, including T cell acute lymphoblastic leukemia.43 D

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Figure 3. Ex vivo vs in vivo Cre-mediated recombination of Notch1 in mouse primary T cells. TAT-Cre (250 nM), alone or in combination with 1.3 μM PTDM, was added to T cells from one Mx1-Cre−/−Notch1f l/f l mouse (black bars). In parallel, T cells were isolated from NOTCH1 KO (Mx1-Cre±Notch1f l/f l) or Control (Mx1-Cre−/−Notch1f l/fl) mice after in vivo treatment with polyI/C (white bars). After 4 h of incubation in RDG medium with 10% FBS, cells were washed and transferred into an anti-CD3ε- and anti-CD28-coated plate for 36 h stimulation. NOTCH1 expression was measured by flow cytometry and is reported as median fluorescence intensity (MFI). Each bar represents the mean + SEM of three independent experiments. **(p < 0.01), ***(p < 0.001) as calculated by one-way ANOVA followed by Tukey’s posthoc test.

Figure 4. CD25 expression after ex vivo or in vivo Cre-mediated Notch1 deletion. TAT-Cre (250 nM), alone or in combination with 1.3 μM PTDM, was added to T cells from a Mx1-Cre−/−Notch1fl/f l mouse (black bars). In parallel, T cells were isolated from NOTCH1 KO (Mx1Cre±Notch1f l/fl) or Control (Mx1-Cre−/−Notch1f l/f l) mice after in vivo treatment with polyI/C (white bars). After 4 h of incubation in RDG medium with 10% FBS, cells were washed and transferred into an anti-CD3ε- and anti-CD28-coated plate for 36 h stimulation. CD25 expression was measured by flow cytometry, and MFI was reported for gated (A) CD4+ and (B) CD8+ T cell subsets. Each bar represents the mean + SEM of three independent experiments. *(p < 0.05), **(p < 0.01), ***(p < 0.001) as calculated by one-way ANOVA followed by Tukey’s posthoc test.

Figure 3 shows NOTCH1 protein levels in stimulated primary T cells, measured by flow cytometry, following ex vivo treatment with TAT-Cre or PTDM/TAT-Cre (black bars) in the presence of 10% FBS, as compared to stimulated untreated cells (Blank). As shown, PTDM/TAT-Cre treatment reduced NOTCH1 protein expression more than 50%, while NOTCH1 protein in the sample treated with TAT-Cre alone was not significantly different from that of the blank 36 h after treatment. These results demonstrate that the PTDM/TATCre system was able to induce loxP recombination both in Rbpjκ and Notch1 floxed primary T cells, suggesting its efficient

gene deletion is not gene-specific. Additionally, equivalent recombination efficiency was observed both in CD4+ and CD8+ T cell subsets, since a comparable level of protein reduction was noted in either CD4+- and CD8+-gated T cells (Figures S5A and S5B, respectively). Notch1 recombination efficiencies obtained with the PTDM/TAT-Cre system ex vivo were also compared to those measured in primary T cells after the in vivo, polyl/C induction of double transgenic mice expressing floxed Notch1 and Cre under the control of the Mx1 promoter (NOTCH1 KO mouse; Mx1-Cre±Notch1f l/fl).21 Remarkably, after treating E

DOI: 10.1021/acsabm.8b00153 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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of using the PTDM/TAT-Cre transduction system ex vivo versus a conventional inducible, tissue-specific transgenic system. The Mx1 promoter requires cytokine production to activate Cre recombinase expression, and these cytokines have inevitable physiological effects on cells, especially on T cells. Using PTDM/TAT-Cre complexes ex vivo, we were able to circumvent any undesired immune cell activation associated with INF-mediated promoter induction, as well as observe a more powerful effect on the expression of downstream proteins associated with deleting our gene of interest. Taken together, these results underscore the superior performance of using PTDM/TAT-Cre over TAT-Cre alone to delete floxed genes in primary T cell ex vivo, as well as compared to conventional in vivo approaches, with the added advantages of achieving greater protein knock-down with less TAT-Cre.

primary T cells ex vivo for just a few hours with PTDM/TATCre (Figure 3, black bar and red line), we achieved an equivalent level of NOTCH1 reduction as obtained by conventional in vivo approaches, whereby NOTCH1 KO mice were given five serial polyl/C injections to induce Cre expression and then rested for 3 weeks (Figure 3, white bar and red dashes). These data highlight the enormous savings of time and mouse-related expenses achieved using PTDM/TATCre to delete genes ex vivo, without sacrificing knock-down efficiency. Downstream Target Expression Following ex Vivo vs in Vivo Notch1 Deletion. CD25, a T cell surface marker, also known as the α-chain of the IL-2 receptor, is upregulated upon T cell activation, and its expression is dependent on NOTCH1 signaling.10,44 Therefore, we would expect CD25 protein expression to be reduced as a consequence of Notch1 deletion prior to T cell stimulation. In the same samples for which we evaluated NOTCH1 expression, we also measured CD25 expression (Figure 4). This allowed us to evaluate whether the level of Notch1 recombination obtained with our ex vivo method was sufficient to control the expression of a downstream target gene. Following the same experimental approach as described previously, we measured CD25 protein expression by flow cytometry. A significant decrease in CD25 expression was observed, separately, on CD4+- and CD8+gated T cells treated with PTDM/TAT-Cre, when compared to untreated cells (Blank) and to cells treated with the same amount of TAT-Cre alone (Figure 4, black bars). We did not observe a significant difference in CD25 expression between Blank and cells treated with TAT-Cre only (Figure 4, black bars). These results mirror Notch1 expression trends observed under the same conditions and suggest that using PTDM/ TAT-Cre to delete Notch1 ex vivo also appreciably reduces the expression of CD25, an important downstream target. We next sought to determine whether the robust reduction of CD25, seen when primary T cells from floxed Notch1 mice were treated ex vivo with PTDM/TAT-Cre (Figure 4, black bars and red line), was also observed when Cre-expressing floxed NOTCH1 mice (NOTCH1 KO, Mx1-Cre±Notch1f l/fl) were treated in vivo with polyl/C (Figure 4, white bars and red dashes). Additional advantages of the ex vivo PTDM/TATCre method over the in vivo polyl/C became evident when we compared CD25 expression levels. While NOTCH1 knockdown caused by PTDM/TAT-Cre generated a measurable downregulation of CD25 expression (black bars), this downstream effect was not observed using in vivo, polyl/Cinduced recombination (white bars), even though Notch1 had been successfully floxed-out (Figure 3, white bars). In fact, after treatment with polyl/C in vivo, followed by a three week rest period to reduce inflammation, there was no significant difference in CD25 expression between NOTCH1KO (Mx1Cre±Notch1f l/f l) and Control (Mx1-Cre−/−Notch1f l/f l) mice (Figure 4, white bars). Moreover, the level of CD25 in the control mice after polyl/C injections (Control, white bar in Figure 4) was significantly higher than in the same transgenic mice that had not been exposed to polyl/C (Blank, black bar in Figure 4), even after the three week recovery time. A likely explanation for this higher, sustained expression of CD25 in polyl/C treated samples is the inflammatory side effects associated with polyl/C. The presence of basal level NOTCH1 at the time when in vivo Cre expression was induced would allow CD25 expression. This discrepancy between ex vivo and in vivo methods highlights another benefit

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CONCLUSIONS Here we provided compelling data that an augmented ex vivo method of delivering TAT-Cre to primary T cells using our synthetic PTDM can overcome the limitations of some inducible transgenic systems for time-specific in vivo gene recombination. We achieved this by treating cells with a combination of the TAT-Cre fusion protein and our PTDM in a noncovalent complex. Due to the catalyzing effect of our PTDM on the transduction efficiency of TAT-Cre, significantly lower amounts of TAT-Cre were required to achieve efficient and specific protein reduction following in vitro and ex vivo Cre-mediated gene deletion. This constitutes a substantial advantage over delivering TAT-Cre alone, considering the challenges and costs associated with producing and purifying fusion proteins,45 as well as toxicity induced by high amounts of TAT-Cre.33 Moreover, delivery to primary T cells is difficult, and the efficiency of PTDM in this cell type offers new valuable opportunities for additional studies. We successfully achieved specific protein reduction, comparable to traditional in vivo methods, without substantial toxic effects to the cells, and in a significantly shortened experimental time frame. Additionally, we could avoid the inflammation and accompanying physiological alterations related to in vivo polyl/ C treatment, making our ex vivo method especially suitable for immunology studies. We further demonstrated the feasibility of this method for the efficient, specific knockout of essential genes of interest in T cell signaling, including Notch1 and Rbpjκ. The resulting reduction in NOTCH1 obtained using PTDM/TAT-Cre was sufficient to affect expression of one of its important downstream targets, CD25, thus demonstrating the applicability of this technology for cell signaling studies and manipulation. Given its significant reduction in cost and time, PTDM/TAT-Cre constitutes a powerful and convenient tool for rapid and efficient screening of gene function in cultured primary mammalian cells expressing floxed genes, supporting and potentiating the discovery of new targets for gene- and cell-based therapies.

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MATERIALS AND METHODS

Cell Culture and PTDM-Mediated TAT-Cre Delivery into the Reporter T Cell Line. GFP-loxP reporter T cells (ABP-RPCGFPLoxT, Allele Biotech) were cultured in RPMI 1640 containing GlutaMAX (Gibco), 1 mM Na pyruvate, nonessential amino acids, 100 U/ml of penicillin, 100 μg/mL of streptomycin, 10 mM HEPES, and 10% FBS. TAT-Cre delivery experiments were carried out in 12F

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well plates. TAT-Cre protein (100 nM) (HTNC; Excellgen, product code RP-7) was mixed with PTDM at increasing molar ratios (2:1; 5:1, and 10:1, PTDM/protein) in PBS (200 μL total volume) and incubated for 30 min at room temperature to favor complex formation. The complex solution was then added dropwise to 2 × 105 cells in serum-free media (1 mL final volume). After 2 h of incubation at 37 °C. the medium was replaced with complete growth medium containing 10% FBS and cultured for an additional 72 h. Gene recombination was analyzed using flow cytometry on 10 000 viable cells (λex 488 nm; λem 530 nm). Cell viability after treatment was determined by double staining with AnnexinV and 7-AAD using the Annexin V PE Apoptosis Detection Kit (Affymetrix, product code 88-8102) by following the vendor’s protocol. Primary Mouse T Cell Isolation and ex Vivo Gene Recombination. Spleens were isolated from mice and manipulated through a 40 μM filter (BD Falcon). Splenocytes were treated with ACK lysis buffer. CD4+ and CD8+ T cells were then isolated using the antimouse CD4+ and CD8+ magnetic particles (BD Pharmingen) and separated using the BD IMag system. Cells (2−3 × 106) were plated in a 1:1 ratio of RPMI-1640 and DMEM medium supplemented with 10% FBS (Gibco), 2 mM L-glutamine, 1 mM Na pyruvate, 100 U/ml penicillin, and 100 μg/mL streptomycin (RDG medium) and treated with a solution of 250 nM TAT-Cre alone or combined with 1.3 μM of PTDM in PBS (complex formed as described above). After 4 h, cells were washed with PBS and plated in normal RDG medium in 12well plates precoated with anti-CD3ε and anti-CD28, which were purified from 145 to 2c11 and 37N hybridoma cell lines, respectively. and cross-linked with anti-Hamster IgG (Sigma). Cells were cultured at 37 °C in a humidified atmosphere with 5% CO2 for 36 h before being analyzed for gene recombination. Flow Cytometry. Samples were surface stained with fluorescentconjugated antibodies: CD4-PerCP, CD25-APC (BD Pharmingen), and CD8-PeCy7 (eBioscience). For intracellular staining, cells were fixed and permeabilized using the Foxp3 staining buffer set (eBioscience) according to the manufacturer’s protocol and incubated with anti-Human/Mouse NOTCH1-PE (eBioscience). Western Blot. Whole cell lysate was made using RIPA buffer (150 mM NaCl, 1% IgeCal-CA 360, 0.1% 619SDS, 50 mM Tris, pH 8.0, 0.5% sodium deoxycholate), and protein concentration was measured by a BCA assay (Thermo Scientific). Thirty μg of total protein lysates were resolved on a 12% SDS-PAGE, transferred to nitrocellulose membrane (Amersham), and blocked in Blotto (5% milk powder, 0.2% Tween-20 in PBS). Membranes were probed overnight with primary antibody, then washed and incubated with horseradish peroxidase (HRP), and labeled secondary antibody. Membranes were developed using ECL reagents (Amersham). Primary antibodies: antiRBPSUH (Cell Signaling) and antiactin (Sigma). Secondary antibodies: antirabbit-HRP, antimouse-HRP (Amersham). Quantification of RBP-Jκ band intensities was carried out by ImageJ (v1.47t) and normalized to actin.

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This work was primarily supported by NSF (CHE-0910963) and UMass Amherst funds. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Mx1-Cre−/−Rbpjκ f l/f l mice were generously provided by Barbara Osborne. Nick Posey and Chris Hango developed Scheme 1.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsabm.8b00153.

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REFERENCES

Synthetic details, flow cytometry histrograms, and dot plots (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Gregory N. Tew: 0000-0003-3277-7925 Author Contributions ∥

F.S., C.S.K., and G.G.-P. contributed equally. G

DOI: 10.1021/acsabm.8b00153 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

Article

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DOI: 10.1021/acsabm.8b00153 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX