Recombineering Linear BACs

Abstract

Recombineering is a powerful genetic engineering technique based on homologous recombination that can be used to accurately modify DNA independent of its sequence or size. One novel application of recombineering is the assembly of linear BACs in E. coli that can replicate autonomously as linear plasmids. A circular BAC is inserted with a short telomeric sequence from phage N15, which is subsequently cut and rejoined by the phage protelomerase enzyme to generate a linear BAC with terminal hairpin telomeres. Telomere-capped linear BACs are protected against exonuclease attack both in vitro and in vivo in E. coli cells and can replicate stably. Here we describe step-by-step protocols to linearize any BAC clone by recombineering, including inserting and screening for presence of the N15 telomeric sequence, linearizing BACs in vivo in E. coli, extracting linear BACs, and verifying the presence of hairpin telomere structures. Linear BACs may be useful for functional expression of genomic loci in cells, maintenance of linear viral genomes in their natural conformation, and for constructing innovative artificial chromosome structures for applications in mammalian and plant cells.

The TelN/tos-assisted precise targeting of chromosome segments (TAPE)

“…5 B). NucleoBond Xtra Midi Plus Kit was also used to extract the linear plasmid with insert segments of 40 kb, 98 kb, 124 kb, 156 kb [34] . The results showed that it was effective to extract the linear plasmid with target DNA sequences of 40 kb and 98 kb, but the DNA band for linear plasmid carrying 124 kb fragment was weak and the linear plasmid carrying the 156 kb fragment could not be recognized ( Supplementary Fig.…”

Phage N15-Based Vectors for Gene Cloning and Expression in Bacteria and Mammalian Cells

“…TelN retains its cleavage-joining activity on the tos sequence to form telomeric ends independent of the N15 replication machinery, regardless of whether TelN was provided in vivo in cis from the tos -containing plasmid or in trans from either of the coresident plasmids in wild-type E. coli (Figure A). Alternatively, tos -containing plasmids can be linearized in vivo in engineered E. coli with chromosomally integrated telN gene (Figure B) ,,, as well as in vitro by recombinant TelN purified from the TelN-expressing E. coli (Figure C). ,, These findings had driven the exploitation of the TelN- tos module as a versatile molecular tool to generate hairpin-capped linear DNA from the standard circular DNA vectors (e.g., plasmids and bacterial artificial chromosome, BACs). , …”

“…Plasmid linearization has been traditionally performed in vitro , either generated with open ends using restriction enzymes , or covalently closed ends via ligating an enzyme-digested DNA fragment with hairpin-forming oligonucleotides . However, restricted DNA with overhang termini is more likely to accumulate damage and be subjected to nuclease attack than end-capped linear DNA. , Unlike the one-step in vivo production of hairpin-capped linear DNA via TelN-expressing E. coli , , these in vitro modifications involve digestion, ligation, and purification steps that contribute to variations in genetic studies while impeding the DNA quality.…”

“…55 However, restricted DNA with overhang termini is more likely to accumulate damage and be subjected to nuclease attack than end-capped linear DNA. 55,56 Unlike the one-step in vivo production of hairpin-capped linear DNA via TelN-expressing E. coli, 14,57 these in vitro modifications involve digestion, ligation, and purification steps that contribute to variations in genetic studies while impeding the DNA quality.…”

“…This feature allows the TelN- tos module to retain its native functions in the heterologous bacterial and mammalian environments. For example, it was experimentally shown that TelN expressed by bacterial and mammalian cells can fully resolve the tos sequence inserted into circular DNA vectors in vitro and in vivo , resulting in linear DNA with telomeric ends. , Second, N15 can replicate extrachromosomally as a linear prophage in E. coli without causing chromosomal integration, displaying one of the essential features required in the design of episomal DNA vectors. Non-N15 plasmids, E. coli chromosome , and human genomic fragments , have been linearized using N15 elements without losing the native gene function or compromising host cell viability.…”