A versatile system for USER cloning-based assembly of expression vectors for mammalian cell engineering

Abstract

A new versatile mammalian vector system for protein production, cell biology analyses, and cell factory engineering was developed. The vector system applies the ligation-free uracil-excision based technique--USER cloning--to rapidly construct mammalian expression vectors of multiple DNA fragments and with maximum flexibility, both for choice of vector backbone and cargo. The vector system includes a set of basic vectors and a toolbox containing a multitude of DNA building blocks including promoters, terminators, selectable marker- and reporter genes, and sequences encoding an internal ribosome entry site, cellular localization signals and epitope- and purification tags. Building blocks in the toolbox can be easily combined as they contain defined and tested Flexible Assembly Sequence Tags, FASTs. USER cloning with FASTs allows rapid swaps of gene, promoter or selection marker in existing plasmids and simple construction of vectors encoding proteins, which are fused to fluorescence-, purification-, localization-, or epitope tags. The mammalian expression vector assembly platform currently allows for the assembly of up to seven fragments in a single cloning step with correct directionality and with a cloning efficiency above 90%. The functionality of basic vectors for FAST assembly was tested and validated by transient expression of fluorescent model proteins in CHO, U-2-OS and HEK293 cell lines. In this test, we included many of the most common vector elements for heterologous gene expression in mammalian cells, in addition the system is fully extendable by other users. The vector system is designed to facilitate high-throughput genome-scale studies of mammalian cells, such as the newly sequenced CHO cell lines, through the ability to rapidly generate high-fidelity assembly of customizable gene expression vectors.

Figure 1. Integration of a GOI into the pBASE backbone vector.

(A) PCR amplification of the GOI using uracil-containing primers and a uracil compatible DNA polymerase (e.g. PfuX7). (B) The PCR fragment of B and a vector backbone fragment obtained by PacI/Nt.BbvCI digest (see Figure S1) is mixed. Addition of the USER™ enzyme mix catalyzes uracil-excision and generates compatible overhangs on the PCR fragment for directed vector assembly. Subsequently, the cloning mix is directly transformed into E. coli cells, where the fragments are ligated together.

Figure 2. Imaging Cell Cytometry of live CHO-S cells transfected with pBASE-vector.

Celigo Imaging Cell Cytometry of live CHO-S cells one day after transfection with (A) pC1_ccdB as negative control, (B) pFAST1-eGFP as positive control, and (C) pBASE2-eGFP as proof of concept for eGFP insertion into the PacI/Nt.BvCI USER cassette of the pBASE2 vector. (A1–C1) cells stained with Hoechst33342. (A2–C2) cytometry of cells with a filter for eGFP fluorescence.

Figure 3. FAST-mediated vector assembly.

Construction of vector types (A–C) requires the same three steps: 1: Preparation of building blocks with appropriate FASTs by PCR or annealing of complementary oligonucleotides. 2: USER fusion and hybridization: the USER enzyme and all building blocks are mixed in one reaction. 3: E. coli transformation with the USER cloning reaction mix. (A) Insertion of a promoter-GOI-terminator expression cassette in an E. coli vector backbone. (B) Assembly illustrated with five elements: the expression cassette as three building blocks, an interchangeable selection marker, and a vector backbone. (C) Similar to (B), but with seven building blocks including C- and N-terminal tags. The N- and C-terminal tag can either be a reporter, a fusion protein, a localization sequence or an epitope tag. GOI: gene of interest; N-tag: N-terminal sequence tag; C-tag: C-terminal sequence tag; P, promoter; and T, terminator.

Figure 4. Confocal laser microscopy of fixed U-2-OS cells transiently transfected with pFAST-vectors.

Confocal laser microscopy of fixed U-2-OS cells transiently transfected with control and pFAST-vectors 48h after transfection with (A) pC1_ccdB as negative control, (B) pFAST1-eGFP, (C) pFAST2-eYFP, (D) pFAST3-eCFP, and (E) pFAST4-mCherry. (A1–E1) microscopy with fluorescence filters. (A2–E2) nuclei stained with DAPI (dark blue). (A3–E3) merged pictures.

Figure 5. Confocal laser microscopy of U-2-OS cells expressing localized fluorescent proteins.

Confocal laser microscopy of fixed U-2-OS cells transiently expressing fluorescent proteins localized to major cellular compartments. Shown are representative images of eYFP or eGFP detected 48 hours after transfection: (A1-3) pFAST6-eYFP::NLS, (B1-3) pFAST5-eGFP::PTS1, (C1-3) pFAST37-eGFP::c-Ha-Ras, (D) pFAST57-CRT::eGFP::KDEL, (E) pFAST58-COXVIII::eGFP, (F) pFAST59-GalNAcT1::eGFP, (G) pFAST61-b1,4GT::eGFP, (H) pFAST56-a-2,6ST-eGFP. (A1–H1) microscopy with fluorescence filters. (A2–H2) nuclei stained with DAPI (dark blue). (A3–H3) merged pictures.