An improved platform for functional assessment of large protein libraries in mammalian cells

Abstract

Multiplex genetic assays can simultaneously test thousands of genetic variants for a property of interest. However, limitations of existing multiplex assay methods in cultured mammalian cells hinder the breadth, speed and scale of these experiments. Here, we describe a series of improvements that greatly enhance the capabilities of a Bxb1 recombinase-based landing pad system for conducting different types of multiplex genetic assays in various mammalian cell lines. We incorporate the landing pad into a lentiviral vector, easing the process of generating new landing pad cell lines. We also develop several new landing pad versions, including one where the Bxb1 recombinase is expressed from the landing pad itself, improving recombination efficiency more than 2-fold and permitting rapid prototyping of transgenic constructs. Other versions incorporate positive and negative selection markers that enable drug-based enrichment of recombinant cells, enabling the use of larger libraries and reducing costs. A version with dual convergent promoters allows enrichment of recombinant cells independent of transgene expression, permitting the assessment of libraries of transgenes that perturb cell growth and survival. Lastly, we demonstrate these improvements by assessing the effects of a combinatorial library of oncogenes and tumor suppressors on cell growth. Collectively, these advancements make multiplex genetic assays in diverse cultured cell lines easier, cheaper and more effective, facilitating future studies probing how proteins impact cell function, using transgenic variant libraries tested individually or in combination.

Figure 1.

Lentiviral Landing Pad (LLP) Derivatives. (A) Schematic of the integration of an attB recombination plasmid into a genomically integrated landing pad. Coding regions are colored boxes, promoters are stemmed arrows, terminator sequences are thick black lines, Bxb1 recombination sequences are stars. (B) LLP constructs, with the original landing pad (AAVS1 LP) for comparison. Components are colored according to panel A, and viral 2A-like sequences are red inverted triangles. Pac is the puromycin resistance gene. Bsd is the blasticidin resistance gene. rtTA is the reverse Tet transactivator. LTR is a lentiviral long terminal repeat. (C) Percentage of BFP positive cells in 293T clones generated with each LLP construct. (D) Recombination rate comparisons of 293T AAVS1 LP clone 4, 293T LLP-Int clone 6 and 293T LLP-Int-Blast clone 2, with a mixture of attB-EGFP and attB-mCherry recombination plasmids without exogenous Bxb1 recombinase expression plasmid (left), or with Bxb1 recombinase expression plasmid included (right). (E) Median fluorescence intensity of each clone from panel C. Left panel shows blue fluorescence before recombination. Right panel shows green fluorescence after recombination of an attB-EGFP recombination plasmid (right). The dotted line represents background fluorescence in parental HEK-293T cells. Panels D and E are a result of at least six replicates.

Figure 2.

Positive or Negative Drug-Based Selection for Recombined Cells. (A) Schematics of two recombination plasmid formats for expressing drug resistance genes. The gray box denotes the coding region for a transgene of interest (PTEN in this analysis). (B) Percentage of red fluorescent cells in mixed populations recombined with the cap-dependent antibiotic resistance vectors, after selection for four or more days in the presence of the indicated concentrations of each drug. Results are an average of three biological replicate experiments, with error bars denoting standard deviation. (C) Schematic of the iCasp9-based negative selection LLP construct. DNA elements are represented in the same manner as Figure 1A. (D) Representative flow cytometry plots of attB-EGFP recombined 293T LLP-iCasp9-Blast Clone 4 cells, before and after negative selection with AP1903. (E) Percent BFP- but GFP+ or mCherry+ positive cells, indicative of recombination, before and after selection with 10 nM AP1903, over eight replicates in 293T LLP-iCasp9-Blast Clone 12.

Figure 3.

A Landing Pad Compatible with Toxic Transgenes. (A) Schematic of the toxic transgene-compatible LLP construct, the corresponding attB recombination vector and an integrated version of this landing pad after insertion of the recombination vector. DNA elements are represented in the same manner as Figure 1A. (B) Representative flow cytometry scatter plots of growth-compatible 293T LLP-rEF1α cells before (top) or after induction with dox (bottom). (C) Scatter plot (top) and smoothed histogram (bottom) of the gated populations in panel B.

Figure 4.

Combinatorial library of tumor suppressors and oncogenes. (A) Schematic showing the combinatorial library construct. The unique plasmid identifier (PID) sequences and representative transgene identifier sequences (GID) are shown. The sequence indicated by the black, bold line was added in a second step after the initial amplification and circularization step, to drive puromycin resistance from the reverse EF1α promoter. The star is the Bxb1 attB site. Black arrows denote direction of coding sequences. (B) Heatmap showing the frequencies of each transgene combination in the final plasmid library.

Figure 5.

Effects of combinatorial library expression on cell proliferation in a mixed culture. (A) Log10-transformed ratio of the frequency of each unique PID sequence associated with each transgene combination in recombined cells divided by the frequency of that PID in the plasmid library. (B) Heatmap showing the mean enrichment or depletion scores of each combination of transgenes after outgrowth. Combinations scored in fewer than 4 replicates are indicated by smaller tiles. Missing combinations are indicated by gray tiles. (C) Histograms of scores from replicate experiments for specific transgene combinations. (D) Enrichment scores of each transgene with all partners. Color indicates the position of the indicated transgene within the 2A-linked translation product. Large hollow points denote the median score for each position, and the black line indicates the mean of the two median points. (E) Heatmap of interaction scores, computed by subtracting each gene's individual median score from the observed growth score for the pair.