Neoantigen-targeted CD8 T cell responses with PD-1 blockade therapy.

Neoantigens are peptides derived from non-synonymous mutations presented by human leukocyte antigens (HLAs), which are recognized by antitumour T cells. The large HLA allele diversity and limiting clinical samples have restricted the study of the landscape of neoantigen-targeted T cell responses in patients over their treatment course. Here we applied recently developed technologies to capture neoantigen-specific T cells from blood and tumours from patients with metastatic melanoma with or without response to anti-programmed death receptor 1 (PD-1) immunotherapy.

Complete motif analysis of sequence requirements for translation initiation at non-AUG start codons.

The initiation of mRNA translation from start codons other than AUG was previously believed to be rare and of relatively low impact. More recently, evidence has suggested that as much as half of all translation initiation utilizes non-AUG start codons, codons that deviate from AUG by a single base. Furthermore, non-AUG start codons have been shown to be involved in regulation of expression and disease etiology.

Evaluation of sgRNA target sites for CRISPR-mediated repression of TP53.

The CRISPR (clustered regularly interspaced short palindromic repeats) platform has been developed as a general method to direct proteins of interest to gene targets. While the native CRISPR system delivers a nuclease that cleaves and potentially mutates target genes, researchers have recently employed catalytically inactive CRISPR-associated 9 nuclease (dCas9) in order to target and repress genes without DNA cleavage or mutagenesis. With the intent of improving repression efficiency in mammalian cells, researchers have also fused dCas9 with a KRAB repressor domain.

Basal p21 controls population heterogeneity in cycling and quiescent cell cycle states.

Phenotypic heterogeneity within a population of genetically identical cells is emerging as a common theme in multiple biological systems, including human cell biology and cancer. Using live-cell imaging, flow cytometry, and kinetic modeling, we showed that two states--quiescence and cell cycling--can coexist within an isogenic population of human cells and resulted from low basal expression levels of p21, a Cyclin-dependent kinase (CDK) inhibitor (CKI). We attribute the p21-dependent heterogeneity in cell cycle activity to double-negative feedback regulation involving CDK2, p21, and E3 ubiquitin ligases.

Quantitative analysis of mammalian translation initiation sites by FACS-seq.

An approach combining fluorescence-activated cell sorting and high-throughput DNA sequencing (FACS-seq) was employed to determine the efficiency of start codon recognition for all possible translation initiation sites (TIS) utilizing AUG start codons. Using FACS-seq, we measured translation from a genetic reporter library representing all 65,536 possible TIS sequences spanning the -6 to +5 positions. We found that the motif RYMRMVAUGGC enhanced start codon recognition and translation efficiency.

Engineering ribosomal leaky scanning and upstream open reading frames for precise control of protein translation.

We have employed upstream open reading frames (uORFs) to systematically tune the translation levels of recombinant proteins. We present the design principles that guided the development of this technology and provide information that may help others in implementing synthetic uORFs for their own applications. We also report on recent applications to our own research projects, including the coupling of uORF and translation initiation site (TIS) engineering with small molecule-inducible post-translational control.

The proliferation-quiescence decision is controlled by a bifurcation in CDK2 activity at mitotic exit.

Tissue homeostasis in metazoans is regulated by transitions of cells between quiescence and proliferation. The hallmark of proliferating populations is progression through the cell cycle, which is driven by cyclin-dependent kinase (CDK) activity. Here, we introduce a live-cell sensor for CDK2 activity and unexpectedly found that proliferating cells bifurcate into two populations as they exit mitosis.

Optimization of oncogene expression through intra-population competition.

Although functional roles have been assigned to many genes, e.g. those involved in cell-cycle regulation, growth signaling, or cancer, considerably less is known about the quantitative relationship between gene expression levels and outcome.

Tuning gene expression with synthetic upstream open reading frames.

We engineered short ORFs and used them to control the expression level of recombinant proteins. These short ORFs, encoding a two-amino acid peptide, were placed upstream of an ORF encoding a protein of interest. Insertion of these upstream ORFs (uORFs) resulted in suppression of protein expression.

Tetracycline-regulated expression implemented through transcriptional activation combined with proximal and distal repression.

Tetracycline-regulated expression systems are widely used to control ectopic gene expression in mammalian cells. However, background or "leaky" expression in the "off" state can limit applications that require control of expression at low levels. In this work we have engineered a tetracycline-regulated expression system with an improved range of control and lower background expression.

Modulating ectopic gene expression levels by using retroviral vectors equipped with synthetic promoters.

Unlabelled: The human cytomegalovirus and elongation factor 1α promoters are constitutive promoters commonly employed by mammalian expression vectors. These promoters generally produce high levels of expression in many types of cells and tissues. To generate a library of synthetic promoters capable of generating a range of low, intermediate, and high expression levels, the TATA and CAAT box elements of these promoters were mutated.

Flow cytometry of v-Abl transformed pre-B cells heterogeneous in ectopic expression levels reveals Ras dose-response.

Flow cytometry is a powerful tool for quantitative biology because it can perform single-cell analysis of large cell populations using multiple parameters. Results are often visualized as a two-dimensional scatter or contour plot. Because these plots can be relatively diffuse, it is not always straightforward to discern a relationship between measured parameters.

Toward a bioengineered heparin: challenges and strategies for metabolic engineering of mammalian cells.

Heparin is the most widely used pharmaceutical to control blood coagulation in modern medicine. A health crisis that took place in 2008 led to a demand for production of heparin from non-animal sources. Since Chinese hamster ovary (CHO) cells are capable of producing heparan sulfate (HS), a related polysaccharide naturally, and heparin and HS share the same biosynthetic pathway, we hypothesized that heparin could be produced in CHO cells by metabolic engineering.

Quantitative assessment of Ras over-expression via shotgun deployment of vectors utilizing synthetic promoters.

We sought to characterize and compare wild-type and oncogenic Ras over-expression. Because different levels of Ras over-expression can have different effects on cell phenotype, it was important to evaluate a wide range of expression. Different expression levels were achieved by using retroviral vectors equipped with different strength promoters.

A cell-compatible conductive film from a carbon nanotube network adsorbed on poly-L-lysine.

Single-walled carbon nanotubes (SWNTs) have shown promise for use in organic electronic applications including thin film transistors, conducting electrodes, and biosensors. Additionally, previous studies found applications for SWNTs in bioelectronic devices, including drug delivery carriers and scaffolds for tissue engineering. There is a current need to rapidly process SWNTs from solution phase to substrates in order to produce device structures that are also biocompatible.

A genetic reporter system to gauge cell proliferation rate.

In higher eukaryotes, E2F transcription factors often drive expression of genes necessary for the cell cycle, notably the G1/S phase transition. With conventional transcriptional reporter systems, expression of a reporter gene from an E2F-responsive promoter would allow one to identify the fraction of cells making this transition. Here, we have engineered an E2F-responsive genetic reporter system that outputs the proliferation rate.

Modular construction of plasmids through ligation-free assembly of vector components with oligonucleotide linkers.

We have developed a modular method of plasmid construction that can join multiple DNA components in a single reaction. A nicking enzyme is used to create 5' and 3' overhangs on PCR-generated DNA components. Without the use of ligase or restriction enzymes, components are joined using oligonucleotide linkers that recognize the overhangs.

Expression of Aire and the early wave of apoptosis in spermatogenesis.

Expression of the autoimmune regulator (Aire) protein in mice and humans is thought to be restricted to the medullary epithelial and monocyte-dendritic cells of the thymus. There it mediates expression and presentation of a large variety of proteins, including those that are peripheral organ-specific and are not expressed by other thymocytes. In this way, self-reactive T lymphocytes that would attack peripheral cells producing these proteins are confronted with the self-Ags and, as a consequence, are deleted.

Activation of an oncogenic microRNA cistron by provirus integration.

Retroviruses can cause tumors when they integrate near a protooncogene or tumor suppressor gene of the host. We infected >2,500 mice with the SL3-3 murine leukemia virus; in 22 resulting tumors, we found provirus integrations nearby or within the gene that contains the mir-17-92 microRNA (miRNA) cistron. Using quantitative real-time PCR, we showed that expression of miRNA was increased in these tumors, indicating that retroviral infection can induce expression of oncogenic miRNAs.

Slow, stochastic transgene repression with properties of a timer.

Background: When gene expression varies unpredictably between genetically identical organisms, this is sometimes ascribed as stochastic. With the prevalence of retroviral vectors, stochastic repression is often observed and can complicate the interpretation of outcomes. But it may also faithfully reflect characteristics of sites in the genome.

AID mutates a non-immunoglobulin transgene independent of chromosomal position.

It is unknown how activation-induced cytidine deaminase (AID) targets immunoglobulin (Ig) genes during somatic hypermutation. Results to date are difficult to interpret: while some results argue that Ig genes have special sequences that mobilize AID, other work shows that non-Ig transgenes mutate. In this report, we have examined the effects of the intronic mu enhancer on the somatic hypermutation rates of a retroviral vector.

Hypermutation rate normalized by chronological time.

It is generally believed that in cells undergoing Ig somatic hypermutation, more cell divisions result in more mutations. This is because DNA synthesis and replication is thought to play roles in the known mechanisms-cytidine deamination and subsequent conversion to thymidine, uracil-DNA glycosylase-mediated repair, mismatch repair, and DNA synthesis by error-prone polymerases. In this study, we manipulated the number of cell generations by varying the rate at which cultures of a mouse cell line were replenished with fresh medium.

Mutational activity in cell line WEHI-231.

The cell line WEHI-231 expresses activation-induced cytidine deaminase (AID), the enzyme that mediates hypermutation and immunoglobulin class switch recombination in activated B cells. Although both the cDNA sequence and protein expression of AID appear normal, the frequency of mutation at the endogenous immunoglobulin locus is low. In this report, we have tested the mutational activity of the cell line with three different indicator constructs.

Directed molecular evolution by somatic hypermutation.

After rearrangement of immunoglobulin gene segments, the immune system evolves the antibody repertoire by mutating the immunoglobulin variable region at a high rate. While this somatic hypermutation was thought to occur only at the variable region, recent studies suggest that hypermutation can occur at locations throughout the genome. Building upon this notion, we sought to exploit this mechanism as a mutagenesis tool.