Synthesis and cell-free cloning of DNA libraries using programmable microfluidics

Microfluidics may revolutionize our ability to write synthetic DNA by addressing several fundamental limitations associated with generating novel genetic constructs. Here we report the first de novo synthesis and cell-free cloning of custom DNA libraries in sub-microliter reaction droplets using programmable digital microfluidics. Specifically, we developed Programmable Order Polymerization (POP), Microfluidic Combinatorial Assembly of DNA (M-CAD) and Microfluidic In-vitro Cloning (MIC) and applied them to de novo synthesis, combinatorial assembly and cellfree cloning of genes, respectively. Proof-of-concept for these methods was demonstrated by programming an autonomous microfluidic system to construct and clone libraries of yeast ribosome binding sites and bacterial Azurine, which were then retrieved in individual droplets and validated. The ability to rapidly and robustly generate designer DNA molecules in an autonomous manner should have wide application in biological research and development

Cell Lineage Analysis of the Mammalian Female Germline

Fundamental aspects of embryonic and post-natal development, including maintenance of the mammalian female germline, are largely unknown. Here we employ a retrospective, phylogenetic-based method for reconstructing cell lineage trees utilizing somatic mutations accumulated in microsatellites, to study female germline dynamics in mice. Reconstructed cell lineage trees can be used to estimate lineage relationships between different cell types, as well as cell depth (number of cell divisions since the zygote). We show that, in the reconstructed mouse cell lineage trees, oocytes form clusters that are separate from hematopoietic and mesenchymal stem cells, both in young and old mice, indicating that these populations belong to distinct lineages. Furthermore, while cumulus cells sampled from different ovarian follicles are distinctly clustered on the reconstructed trees, oocytes from the left and right ovaries are not, suggesting a mixing of their progenitor pools. We also observed an increase in oocyte depth with mouse age, which can be explained either by depth-guided selection of oocytes for ovulation or by post-natal renewal. Overall, our study sheds light on substantial novel aspects of female germline preservation and development

Computer-aided high-throughput cloning of bacteria in liquid medium

Bacterial cloning was first introduced over a century ago and has since become one of the most useful procedures in biological research, perhaps paralleled in its ubiquity only by PCR and DNA sequencing. However, unlike PCR and sequencing, cloning has generally remained a manual, labor-intensive, low-throughput procedure. Here we address this issue by developing an automated, computer-aided bacterial cloning method using liquid medium that is based on the principles of (i) limiting dilution of bacteria, (ii) inference of colony forming units (CFUs) based on optical density (OD) readings, and (iii) verification of monoclonality using a mixture of differently colored fluorescently labeled bacteria for transformation. We demonstrate the high-throughput utility of this method by employing it as a cloning platform for a DNA synthesis process

GMP-like and MLP-like Subpopulations of Hematopoietic Stem and Progenitor Cells Harboring Mutated EZH2 and TP53 at Diagnosis Promote Acute Myeloid Leukemia Relapse: Data of Combined Molecular, Functional, and Genomic Single-Stem-Cell Analyses

Acute myeloid leukemia (AML) is associated with unfavorable patient outcomes primarily related to disease relapse. Since specific types of leukemic hematopoietic stem and progenitor cells (HSPCs) are suggested to contribute to AML propagation, this study aimed to identify and explore relapse-initiating HSPC subpopulations present at diagnosis, using single-cell analysis (SCA). We developed unique high-resolution techniques capable of tracking single-HSPC-derived subclones during AML evolution. Each subclone was evaluated for chemo-resistance, in vivo leukemogenic potential, mutational profile, and the cell of origin. In BM samples of 15 AML patients, GMP-like and MLP-like HSPC subpopulations were identified as prevalent at relapse, exhibiting chemo-resistance to commonly used chemotherapy agents cytosine arabinoside (Ara-C) and daunorubicin. Reconstruction of phylogenetic lineage trees combined with genetic analysis of single HSPCs and single-HSPC-derived subclones demonstrated two distinct clusters, originating from MLP-like or GMP-like subpopulations, observed both at diagnosis and relapse. These subpopulations induced leukemia development ex vivo and in vivo. Genetic SCA showed that these relapse-related subpopulations harbored mutated EZH2 and TP53, detected already at diagnosis. This study, using combined molecular, functional, and genomic analyses at the level of single cells, identified patient-specific chemo-resistant HSPC subpopulations at the time of diagnosis, promoting AML relapse

Retrospective cell lineage reconstruction in Humans using short tandem repeats

Cell lineage analysis aims to uncover the developmental history of an organism back to its cell of origin1. Recently, novelin vivomethods and technologies utilizing genome editing enabled important insights into the cell lineages of animals2–8. In contrast, human cell lineage remains restricted to retrospective approaches, which still lack in resolution and cost-efficient solutions. Here we demonstrate a scalable platform for human cell lineage tracing based on Short Tandem Repeats (STRs) targeted by duplex Molecular Inversion Probes (MIPs). With this platform we accurately reproduced a known lineage of DU145 cell lines cells9and reconstructed lineages of healthy and metastatic single cells from a melanoma patient. The reconstructed trees matched the anatomical and SNV references while adding further refinements. Our platform allowed to faithfully recapitulate lineages of developmental tissue formation in cells from healthy donors. In summary, our lineage discovery platform can profile informative STR somatic mutations efficiently and we provide a solid, high-resolution lineage reconstruction even in challenging low-mutation-rate healthy single cells.</jats:p

Lack of lineage barriers between oocytes sampled from the left and right ovary suggests a spatially incoherent mode of expansion of primordial germ cells during embryonic development.

<p>a) Two modes of expansion-migrations of a small pool of progenitors which become lineage restricted during the epiblast stage. In a spatially coherent mode (1) progenies remain physically close together as they expand in numbers and thus separation into the two ovaries creates two distinct populations. In a spatially incoherent mode (2) progenies physically mix and thus separation into the two ovaries result in equal samples of the original progenitors. b) Reconstructed cell lineage trees show that GV oocytes from large antral follicles of the right (blue) or left (red) ovaries do not form distinct clusters. As a positive control cumulus cells sampled from two follicles – one in the right ovary (green) and one in the left (purple) do form distinct clusters, as expected given the previously estimated small number of progenitors of the epithelial population of a given follicle.</p

Depth-guided oocyte selection for ovulation accounts for the increase in oocyte depth with age.

<p>(a) Depth-guided oocyte maturation. Shown is a hypothetical tree showing entry into meiosis during embryonic development. The hypothesis states that oocytes that enter meiosis first during embryonic life would be ovulated first during post-natal life. (b) Comparison of median depth of oocytes isolated from pre antral follicles isolated from 12 day-old mice vs. oocytes isolated pre ovulatory follicles isolated from older mice. Each point is the median of the depths of all oocytes sampled from a single mouse. (c) Depth distributions of oocytes from pre-antral follicles in 12 day-old mice are not significantly different from the expectation from the production line hypothesis. Shown is the expected distribution of median depth of oocytes assuming that the depth distribution at birth is a uniform average of all measured post-natal depth values of maturing oocytes. Filled circles denote the observed median depth in the two 12 day old mice. Error bars are standard errors of the means.</p

Oocyte depth increases with age.

<p>a–c) Reconstructed lineage trees of GV oocytes from large antral follilces in three mice demonstrate an increase in oocyte depth (Y axis) with age. The putative zygote is at depth 0. Median depth of oocytes is 13 divisions in M27, a 27 day old mouse (Mouse names represent their age in days) (a), 16 divisions in mouse M159 (b) and 19 in mouse M268 which contains 6 ovulated oocytes which were marked on the tree with blue dots (c). Horizontal red lines denote the median depth. All lineage trees were reconstructed using the maximum-likelihood neighbor joining method and rooted with the median identifier of all cells. d) Median depth of GV oocytes from large antral follicels increases with age. Each blue point is the median of the depths of all oocytes sampled from a single mouse. The Pearson correlation of median depth and age is 0.81 (p = 0.007). High inter-mouse variability can be seen. Errorbars are standard errors of the means. (e) Depth of pancreatic islet cells does not increase with age. Shown are the depths of pancreatic islet cells from M36, a 1-month old mouse and from M280, a 9-month old mouse.</p