Transient AID expression for in situ mutagenesis with improved cellular fitness

Abstract

Activation induced cytidine deaminase (AID) in germinal center B cells introduces somatic DNA mutations in transcribed immunoglobulin genes to increase antibody diversity. Ectopic expression of AID coupled with selection has been successfully employed to develop proteins with desirable properties. However, this process is laborious and time consuming because many rounds of selection are typically required to isolate the target proteins. AID expression can also adversely affect cell viability due to off target mutagenesis. Here we compared stable and transient expression of AID mutants with different catalytic activities to determine conditions for maximum accumulation of mutations with minimal toxicity. We find that transient (3-5 days) expression of an AID upmutant in the presence of selection pressure could induce a high rate of mutagenesis in reporter genes without affecting cells growth and expansion. Our findings may help improve protein evolution by ectopic expression of AID and other enzymes that can induce DNA mutations.

Figure 1

Constructs and screening system. (A) 293FT/RFP1 cells stably express RFP1 fluorescent protein. RFP1 florescence loss after AID expression is used to assess AID mutagenic activity. (B) Schematic representation of the AID expression vector. A CMV promotor is followed by a human AID or AID mutant gene which is linked to an HA tag at the C-terminus followed by furin/2 A peptide (F2A) bicistronic expression linker and an eGFP reporter gene. An internal ribosome entry site (IRES) is used for bicistronic expression of a puromycin resistance gene. (C) Cell lysates from 293FT/RFP1 cells expressing AID mutants were used to perform immunoblot analysis with antibodies binding to the HA tag on AID or tubulin as a cell loading control.

Figure 2

Stable AID expression induces a spike of RFP fluorescence loss. 293FT/RFP1 cells were stably transduced with AID mutants and then 10⁵ cells were analyzed for somatic hypermutation by RFP fluorescence loss using a flow cytometer (A) 10 days after stable expression or (B) periodically over one month. The results show the mean values of three replicates ± S.D. Significant differences between RFP loss after expression of S38A or m7.3 mutants compared to AID-WT are indicated; **P ≤ 0.01.

Figure 3

Optimization of transient transfection for efficient somatic hypermutation. (A) Comparison between expression of AID m7.3 in HEK293FT and HEK293 cells over time. Cells transiently transfected with m7.3 plasmid were collected every 2 days for 6 days. Cell lysates were used to perform immunoblot analysis with antibodies against the HA tag on AID or tubulin as a cell loading control. (B) Comparison between AID expression level after stable and transient transfection. Cells were transfected stably or transiently with m7.3 AID. Cell lysates prepared after 3 and 7 days were used to perform immunoblot analysis with antibodies binding to the HA tag on AID or tubulin as a cell loading control. (C) Mean fluorescence intensity of eGFP in 10⁵ cells measured at day 7 including 3 days with or without puromycin selection, n = 3; Bars, S.D. (D) Cells transiently transfected with AID plasmids and selected with puromycin (2 µg/mL) for 3 days. Cell lysates prepared at 7 and 14 days after transient transfection were used to perform immunoblot analysis with antibodies binding the HA tag on AID or tubulin as a cell loading control. (E) Reporter cells (293FT/RFP1) were transfected with different AID mutants and selected for 0, 2 or 4 days with 2 µg/mL puromycin. RFP loss was measured 10 days after transient transfection. (F) RFP loss with or without puromycin selection. Results show mean frequencies of RFP loss measured in 10⁵ cells at each time (n = 3). Significant differences between mean values are indicated; **P ≤ 0.01, ***P ≤ 0.001, ns, not significant.

Figure 4

Robust RFP1 loss by transient expression of AID. (A) 293FT/RFP1 cells transiently transfected with plasmids encoding AID mutants, selected with puromycin for 3 days and analyzed for somatic hypermutation by RFP fluorescence loss. Results show mean values of three experiments ± SD. (B) Representative flow cytometry results and (C) analysis of accumulative RFP loss after one and two rounds of transient AID expression. Results show the mean values of three replicates ± SD. Significant differences between RFP loss after the first and the second AID transfections are indicated; **P ≤ 0.01, ***P ≤ 0.001.

Figure 5

Robust DsRed reversion by transient AID expression. (A) 293FT/DsRed2s reporter cells stably express DsRed with a premature stop codon within an AID hotspot motif. AID expression can remove the premature stop codon and restore DsRed florescence. (B) 293FT/DsRed2s cells stably transduced with AID mutants (10⁶ cells) were analyzed for gain of red florescence over time. n = 3; Bars, SD. (C) 293FT/DsRed2s cells transiently transfected with AID mutants and selected with puromycin for 3 days (10⁶ cells) were analyzed for gain of red florescence over time. n = 3; Bars, SD. (D) Frequency of mutations in the DsRed2 gene obtained by PCR amplification from unsorted cells 14 days after expression of AID mutants stably or (E) transiently with puromycin selection. Bars, SEM. (F) Pie charts illustrating the number of mutations in PCR-amplified sequences from unsorted cells 14 days after expression of AID mutants stably or (G) transiently. The number in the middle of the pie chart indicates the total number of sequences analyzed. Below the pie charts, mutation matrix shows nucleotide substitution pattern identified from sequencing results.

Figure 6

Transient expression of AID reduces its adverse effect on cell growth. Growth curve of HEK293FT cells (A) virally transduced with AID mutants or (B) transiently transfected with AID expressing plasmids. Results show mean values ± SD (n = 3). (C) Western blots show AID expression in cell extracts from 3T3 and 3T3 TetOn-AID cells after 4 days of culturing the cells with or without 0.5 µg/mL doxycycline; tubulin was used as loading control. (D) AID induction as confirmed by bicistronic GFP expression in 10⁵ cells of each 3T3 and 3T3 TetOn-AID cells after 4 days of culturing the cells with or without 0.5 µg/mL doxycycline. (E) Growth curve of 3T3 and 3T3 TetOn-AID cells with or without 0.5 µg/mL for 6 days. Results show mean values ± SD (n = 3). Significant differences between growth curves are indicated; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. ns, not significant.