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    Lentiviral Barcode Libraries for Cell Labeling and Clonal Tracking

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    The ability to label and trace individual cells is a powerful experimental approach in many research areas including stem cell biology, development, evolution, and carcinogenesis. While numerous approaches exist for individual cell labeling, the widespread accessibility of next-generation sequencing (NGS) technology provides a new approach for tracking clonal variations in large cell populations. Using libraries of diverse sequences in lentiviral vectors, such as Cellecta’s CloneTracker™ Lentiviral Barcode Libraries, it is possible to genomically label each cell in a population of a million with a uniquely identifiable short nucleotide sequence (i.e., a barcode).

    The initial transduction of a lentiviral barcode library into cultured cells produces a founder or starter population in which each cell has a unique barcode label. Since the barcodes genomically integrate, they are heritable so all progeny from each cell contain the same sequence and clonal expansion for each founder cell can be monitored.

    Assess clonal heterogeneity by NGS analysis of barcodes

    This labeling of target cells with clonal barcodes using lentiviral libraries offers an effective approach for monitoring cell phenotypes in a time-course experiment. Since all progeny of the starter cells inherit barcodes from their progenitors, changes in the heterogeneity of the barcode distribution over time directly correspond to how growth conditions, differentiation, or selection affect survival and growth of each clone in the original cell population.  For example, the figure below shows as experiment where barcode representation was used to investigate differences in how the same cells grow and expand under typical in vitro cell culture as compared to when they form a tumor after engraftment into a mouse.

    Depending on the construction of the library vector, a clonal barcode construct can be constructed so that that the barcode sequence can not only be read from genomic DNA but also expressed so it is detectable by sequencing the RNA cell fraction. For example, Cellecta offers barcode libraries (i.e., CloneTracker XP™ Libraries) where the barcode is located in the 3’-UTR of the antibiotic selection gene for the vector, as shown in the figure.  With this configuration, the clonal barcodes can be detected in single cell bead-based RNA expression profiling assays, such as 10X Genomics System, BD Rhapsody, or Dolomite Bio’s Nadia system, so that changes in gene activation in different clonal populations can be assayed over the course of an experiment to identify sub-populations of progeny with advantageous or harmful phenotypes relative to the specific conditions, such as increased drug resistance or sensitivity.

    Studies to understand how activation or deactivation of specific genetic pathways generates phenotypic changes of interest in different clonal populations can also be taken a step further by actually adding gene effectors, such as a CRISPR sgRNAs, to barcode libraries designed for RNA expression. The sgRNA (or shRNA) elements act to disrupt expression of their targeted gene which purposefully introduces a specific genetic perturbation into the cell. With these libraries, then, cells from functional genetic screens to identify genes required for specific phenotypes or essential for viability, can also be analyzed at a single-cell level to assess how knockout or particular genes affects gene regulation and pathway activation in the same cells.

    While this sort of assay can be done with standard, non-barcoded sgRNA libraries as long as the sgRNA sequence itself is expressed on a poly-A transcript that can be detected by RNA sequencing, the addition of clonal barcodes with the sgRNA provides an index to demonstrate similar responses in multiple examples of cells that receive the same sgRNA. These library barcodes combined with sgRNA are sometimes referred to as Universal Molecular Indicators (UMIs) since they provide a unique barcode for each instance a particular sgRNA sequence is ligated into the library. As a result, when the same sgRNA is introduced into multiple cells during transduction at the start of a CRISPR screen, each cell that picks up the sgRNA is separately labeled with a different barcode. At the end of the screen then, each clonal population from each transduction of a particular sgRNA is independently identifiable.

    Clonal barcode labeling of cells using complex lentiviral libraries of uniquely identifiable barcodes offers a way to analyze cell heterogeneity and expansion in a range of experimental contexts. These tools can help investigators better understand a diverse range of phenomena, including:

    • how cell subpopulations with advantageous phenotypes develop and thrive
    • how growth conditions affect cell heterogeneity or clonal subpopulations
    • what factors influence survival rate and expansion of tumors
    • how progenitor cell populations differentiate, and
    • what contributes to the expansion of cancer cells with various genetic driver lesions or drug tolerances.

    The combination of NGS with cell barcode labeling has the potential to vastly increase our understanding of cell growth, differentiation, pathogenicity, and drug resistance.

    More information on Cellecta’s CloneTracker XP-CRISPR Human and Mouse Lentiviral Barcode library offerings available here.

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