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The development of chromatin immunoprecipitation methods coupled with DNA microarray (ChIP-chip) technology has enabled genome-wide identification of cis-DNA regulatory elements to which transcription factors bind. Nonetheless, the ChIP-chip technology requires antibodies with extremely high affinity and specificity for the target transcription factors. Unfortunately, such antibodies are not available for most human transcription factors. In principle, this problem can be circumvented by utilizing ectopically expressed epitope-tagged proteins recognizable by well-characterized antibodies. However, such expression is no longer endogenous. To surmount this problem, we have successfully developed a facile method to knock-in a 3xFlag epitope into the endogenous gene loci of transcription factors. The knock-in approach provides a general solution for the study of proteins for which antibodies are substandard or not available.
The human genome encodes approximately 25,000 proteins. Characterizing all 25,000 depends on availability of high quality antibodies that can be used for multiple applications including Western blot, immunofluorescence (IF), and immunoprecipitation (IP). For analysis of transcription factors and other DNA-binding proteins, “ChIP grade” antibodies capable of immunoprecipitating the protein of interest within the context of chromatin are most often desired . Notwithstanding, ChIP-grade antibodies exist for only a small fraction of chromatin-associated proteins. This is particularly problematic for ChIP-chip or ChIP-sequencing studies, where the use of more than one antibody is highly recommended. The antibody problem can be circumvented by generating cell lines that stably express epitope-tagged proteins recognizable by available antibodies, but this approach is far from ideal given that expression is no longer endogenous, which may complicate interpretation of results. Moreover, the construction of recombinant plasmids containing both full length cDNA and epitope sequences can be cumbersome, particularly for proteins encoded by large transcripts.
Epitope-tagging by homologous recombination-mediated knockin (KI) is an effective means for biochemical and cellular studies of proteins in recombination prone organisms, such as yeast . Applying this approach to somatic mammalian cells is not feasible due to low frequency of homologous recombination between exogenous plasmid and specific genomic loci. Recent studies have shown that this problem can be circumvented by delivering constructs with recombinant adeno-associated virus (rAAV), which can increase the frequency of homologous recombination to as much as 2% . We have successfully developed a method whereby rAAV is used to “knock in” epitope tag sequences into targeted loci in human somatic cells . The tagged proteins, which harbor three Flag epitopes in tandem (3xFlag), can be exploited for Western blot, IP, IF, and ChIP-chip analyses . Here, I describe step-by-step protocol of the 3xFlag knockin approach.
The 3xFlag tag sequences are inserted before the stop codon of target genes through rAAV-mediated homologous recombination (outlined in Figure 1). The entire procedure can be arbitrarily divided into 6 major steps: (1) Targeting vector construction; (2) rAAV targeting virus generation; (3) Gene targeting of human cells; (4) Genomic DNA preparation; (5) Targeted clone screening; and (6) Excision of the Neomycin resistance gene. It takes ~ 45 days to generate 3xFlag knock-in clones in DLD1 cells.
We also developed a one-step highly efficient targeting vector construction strategy (Figure 2). Recently, the New England Biolabs has developed the USER (uracil-specific excision reagent) cloning technique, which facilitates assembly of multiple DNA fragments in a single reaction by in vitro homologous recombination and single-strand annealing . In this system, the vector contains a cassette with two inversely oriented nicking endonuclease sites separated by restriction endonuclease site(s). The vector is then digested and nicked with restriction endonucleases, yielding a linearized vector with 8-nucletide single-stranded, non-complimentary overhangs. To generate target molecules for cloning into this vector, a single deoxyuridine (dU) residue is placed 8 nucleotides from the 5′-end of each PCR primer. In addition to the dU, the PCR primers contain sequence that is compatible with each unique overhand on the vector. After amplification, the dU is excised from the PCR products with a uracil DNA glycosylase and an endonuclease (the USER enzyme), generating PCR products flanked by 3-prime, 8 nucleotide single-stranded extensions that are complementary to the vector overhangs. When mixed together, the linearized vector and PCR products directionally assemble into a recombinant molecule through complementary single-stranded extensions. To make the rAAV-mediated targeting vector compatible with the USER cloning system, we inserted cassette A (Cst A) between L-ITR and 3xFlag sequences, and cassette B (Cst B) between the right lox P site and R-ITR of the AAV-3xFlag knockin vector to generate the AAV-USER-3xFlag-KI vector (Figure 2). These cassettes contain two inversely oriented nicking endonuclease sites (Nt. BbvCI) separated by restriction endonuclease sites (Xba I). After treatment with Nt.BbvC I and Xba I restriction enzymes, the AAV-USER-3xFlag-KI vector is digested into a 3xFlag-lox P-Neo-lox P fragment flanked by two 5′ single-stranded overhangs (Figure 2) and a vector backbone flanked by two 5′ overhangs (Figure 2). PCR is then used to amplify left and right homologous arms from genomic DNA. The sequence GGGAAAGdU is added to the 5′ of the forward left arm primers, and GGAGACAdU is added to the reverse left arm primers. GGTCCCAdU is added to the forward right arm primers and GGCATAGdU to the reverse left arm primers. The PCR products are then treated with the USER enzymes to generate single-stranded overhangs. Finally, the left and right arms are mixed with the two vector fragments followed by bacterial transformation (Figure 2).
|Forward primer:||add GGGAAAGdU to the 5′ end of the designed PCR primer.|
|Reverse primer:||add GGAGACAdUnn to the 5′ end of the reverse sequences of the upstream of stop codon (the first n could be A, T, G, or C; the second n could be any nucleotides but A so that the 3xFlag is in frame fused with the targeted gene, and avoid to introduce a stop codon before the 3xFlag).|
|Forward primer:||add GGTCCCAdU to the downstream sequences of stop codon.|
|Reverse primer:||add GGCATAGdU to the 5′ end of the designed PCR primer.|
|10 μl reaction||1||15|
|10 × HiFi Buffer||1||15|
|Primer 1 (50μM)||0.06||0.9|
|Primer 2 (50μM)||0.06||0.9|
|HifiTaq (5 u/μl)||0.1||1.5|
|10 × Buffer||1||13||26||50||75||102|
|Primer 1 (50 μM)||0.06||0.78||1.56||3||4.5||6.12|
|Primer 2 (50 μM)||0.06||0.78||1.56||3||4.5||6.12|
The author would like to thank Dr. Chao Wang for proof reading. This work was supported by RO1 CA127590 and RHG004722A.