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Isolation of rare recombinants without using selectable markers for one-step seamless BAC mutagenesis

Nature Methods volume 11, pages 966–970 (2014)Cite this article

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Abstract

Current methods to isolate rare (1:10,000–1:100,000) bacterial artificial chromosome (BAC) recombinants require selectable markers. For seamless BAC mutagenesis, selectable markers need to be removed after isolation of recombinants through counterselection. Here we illustrate founder principle–driven enrichment (FPE), a simple method to rapidly isolate rare recombinants without using selectable markers, allowing one-step seamless BAC mutagenesis. As proof of principle, we isolated 1:100,000 seamless fluorescent protein–modified Nodal BACs and confirmed BAC functionality by generating fluorescent reporter mice. We also isolated small indel P1 phage–derived artificial chromosome (PAC) and BAC recombinants. Statistical analysis revealed that 1:100,000 recombinants can be isolated with <40 PCRs, and we developed a web-based calculator to optimize FPE.

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Figure 1: Seamless BAC recombineering via the conventional approach and FPE.
Figure 2: Conceptual basis of FPE.
Figure 3: Functional integrity of fluorescent protein–modified Nodal BACs isolated by FPE.
Figure 4: Cost function analysis of FPE for high-throughput applications.

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Acknowledgements

We acknowledge S.K. Sharan (US National Cancer Institute) for providing mini-λ and for helpful discussions; M.L. Condic, F.S. Gizatullin, J.T. Hill and R.A. McKnight for comments on the manuscript; V. Lyozin and B. Stephan for technical help in figure preparation; and V. Boyko (US National Cancer Institute) for providing plasmids containing the coding sequences of fluorescent proteins. This work was supported by funds from the University of Utah School of Medicine's Department of Pediatrics (Neonatology) and an American Heart Association grant to L.B. (09BGIA2251076); the US National Heart, Lung, and Blood Institute Bench-to-Bassinet Consortium grant to H.J.Y. (U01HL0981); and the Intramural Research Program of the US National Institutes of Health and National Cancer Institute to M.R.K. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services; nor does mention of trade names, commercial products or organizations imply endorsement by the US government.

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Author notes

  1. Paul C Bressloff and Amit Kumar: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Pediatrics, University of Utah School of Medicine, Salt Lake City, Utah, USA

    George T Lyozin, Yasuhiro Kosaka, H Joseph Yost & Luca Brunelli

  2. Department of Mathematics, University of Utah, Salt Lake City, Utah, USA

    Paul C Bressloff

  3. Laboratory of Protein Dynamics and Signaling, National Cancer Institute, Frederick, Maryland, USA

    Amit Kumar & Michael R Kuehn

  4. Department of Neurobiology and Anatomy, University of Utah School of Medicine, Salt Lake City, Utah, USA

    Bradley L Demarest & H Joseph Yost

Authors
  1. George T Lyozin
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Contributions

G.T.L. and L.B. developed the project; G.T.L. and Y.K. performed experiments; P.C.B. carried out the mathematical analysis; A.K. analyzed mice; M.R.K. directed mouse research and provided editing; B.L.D. developed the web calculator; H.J.Y. provided research support, resources and editing; L.B. and G.T.L. wrote the manuscript; L.B. directed the overall project.

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Correspondence to Luca Brunelli.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Generation of markerless targeting vectors.

A single PCR was used to generate markerless targeting vectors, i.e. including only the 720-nucleotide coding sequence of EYFP and the ~80-nucleotide homology arms flanking the first Nodal methionine. The PCR products of a few PCR tubes were combined (making up ~200 μl PCR mix) and treated as described in Methods; then precipitated with isopropanol and dissolved in 30 μl deionized water. 1 μl DNA solution (containing ~75 ng DNA) was loaded in lane1. Lane2: 1 kb plus DNA ladder. A similar approach was used for mStrawberry.

Supplementary Figure 2 Diagram of the FPE procedure.

The cell numbers used as examples are the same as those used in the Result section. The steps in the procedure are numbered one to ten. SC, saturated culture; DC, diluted culture; DDC, diluted and divided culture. Red circles indicate positive cultures. See Result for details.

Supplementary Figure 3 Daily work schedule for the FPE procedure.

Each working day includes the end of the current FPE cycle, i and the beginning of the next enrichment cycle, i+1 (when required). It starts by doing PCR on SCs (1a) and, if a positive culture is detected, performing step 1b to calculate the number of total cells in DDC (Ni) so that the recombinant frequency fi in the positive SCs (fi = 1/Ni) can be estimated. If no positive culture is detected, steps 4a and 4b need to be repeated using more cells. Once fi is calculated, compare it with the target frequency of recombinants (fT) to determine whether enrichment is adequate or another enrichment cycle is needed (Supplementary Fig. 4). If a new enrichment cycle is needed, perform steps 3, 4a, and 4b to establish DCs as well as DDCs of the next enrichment cycle, which concludes the working day. Crossed red circles indicate positive cultures.

Supplementary Figure 4 Workflow for the FPE procedure.

The aliquot taken from the electroporation mix should contain at least one recombinant, i.e. n0,D ≥1 among N0,D total cells surviving the electroporation shock. After dilution and division of the aliquot, every positive parental SC is independent, i.e. composed of recombinant cells originating from different recombination events. Diamond shape: the target frequency (fT) is the recombinant frequency sufficient to isolate recombinants by PCR screening individual clones transferred from agar plates and grown in 96-well plates or PCR tube strips (ideally in the range of 1/1 to 1/40). LM: liquid medium. D, diluted. DD, diluted and divided. S, saturated. For details on subscripts, see Methods and Supplementary Note.

Supplementary Figure 5 Recombinants in reconstituted cell mixes can be detected with high sensitivity.

(a) Bacterial cultures containing wild type and EYFP Nodal BACs were grown separately. The wild type culture was diluted to 109 cells/ml. The EYFP culture was diluted into the wild type culture to produce different ratios of EYFP:wild type Nodal BAC containing cells. 1 μl cell mix (containing about 106 totals) was used for PCR in a 10 μl reaction mix. PCR was run for 40 cycles and all products were loaded on agarose gel. Lane one: 103 recombinants:106 totals. Lane two: 102 recombinants:106 totals. Lane three: 10 recombinants:106 totals. Lane 4: 1 recombinant:106 totals. (b) The cultures and PCRs were prepared as above, but the wild type culture was concentrated 10-fold in 5 mM MgSO4 to increase the titer to 1010 cells/ml. Here, 1 μl cell mix contained about 107 totals. PCR was run for 45 cycles. Lane one: 102 recombinants:107 totals. Lane two: 10 recombinants:107 totals. Lane three: 1 recombinant:107 totals. Note that 106 (panel a, lane four) and 107 (panel b, lane three) are the maximal, theoretical sensitivity of the assay, and approaching this limit leads to genetic drift. This means that some samples will receive one cell, others more than one, and some no cell from the same 10-6 to 10-7 cell mixes. This implies that a few PCR with different replicas of the same culture might be needed when working with recombinant frequencies close to the theoretical sensitivity of the assay.

Supplementary Figure 6 Recombinant frequencies in DDCs and the corresponding SCs coincide.

Data were obtained from Supplementary Tables 1 and 2. The recombinant frequency in DDCs was estimated as the reciprocal of the number of founders establishing the DDC, while the recombinant frequency in SCs was estimated as the fraction of recombinant clones obtained by plating an appropriately diluted aliquot of SCs on agar. The two-sided 95% confidence interval of each recombinant frequency in each experiment was calculated using the Clopper-Pearson exact method. For all PACs and BACs, the confidence interval of DDC and SC recombinant frequencies showed broad overlap and thus the null hypothesis, i.e. there is no growth difference, could not be rejected.

Supplementary Figure 7 Recombinants and nonrecombinant BACs grow similarly.

Using 100 and 120 min as time points, the growth rates between these time points can be calculated according to the formula:

The difference (Δ) between growth rates in nonrecombinants and recombinants are: ΔRP24-64L14 = -0.00432; ΔRP24-204A9 = -0.00139; ΔRP23-15D8 = 0.001486. Negative values indicate that recombinants grow faster than nonrecombinants. Rec: recombinant BAC; Non-Rec: nonrecombinant BAC. OD: optical density at 600 nm.

Supplementary Figure 8 The majority of saturated parental cultures are positive for recombinants.

EYFP targeting DNA was electroporated into recombineering electrocompetent cells as described in Methods. An aliquot of the electroporation mix was diluted and plated in a 96-well plate. Cultures were grown and genotyped for the 5' break-point. All culture PCR genotyping products were loaded in gel, but only 48 are shown. Arrows indicate PCR products.

Supplementary Figure 9 Calculating the recombination frequency using parental cultures.

A colony containing RP24-204A9(-) BAC (Supplementary Table 2) and mini-lambda was used to prepare electro- and recombineering competent cells. A 200-nucleotide oligonucleotide (oligo ID #11, Supplementary Table 3) with FRT, LoxP, and KpnI sites flanked by 63-nucleotide homology arms was electroporated into cells. An aliquot of the electroporation mix was diluted and divided in a 96-well plate so that parental cultures were established with 25 cells on average. After DDCs reached saturation, SCs were screened with primers (oligo IDs #29 and #30, Supplementary Table 3) as shown in this figure. The proportion of negative cultures was used to estimate the frequency of recombination as previously described1. The number of negative cultures was 38, the proportion of negative cultures was about 0.4, and the negative natural logarithm of the proportion (-ln 0.4) was 0.92, implying that parental cultures received on average 0.92 recombinants every 25 total cells from the recombineering mix, i.e. the recombination frequency is 0.037 or 3.7%. We similarly estimated recombination frequency in parental cultures established with seven and three total cells, and found the frequency of recombination to be 3.7% and 3% (Supplementary Table 2), respectively. A positive parental culture from the ones established with three total cells was used to isolate recombinants.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9, Supplementary Tables 1 and 2 and Supplementary Note (PDF 2042 kb)

Supplementary Table 3

Targeting single-stranded and priming double-stranded marker oligonucleotides and corresponding detecting primers (XLSX 15 kb)

Supplementary Software

(http://yost.genetics.utah.edu/software.php) Founder Principle-Driven Enrichment Calculator. ZIP file of the computer code used to generate the cost function analysis web calculator (ZIP 7 kb)

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Lyozin, G., Bressloff, P., Kumar, A. et al. Isolation of rare recombinants without using selectable markers for one-step seamless BAC mutagenesis. Nat Methods 11, 966–970 (2014). https://doi.org/10.1038/nmeth.3030

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