Navigation Links
Efficient Cloning and Electro-transformation of Large Eukaryotic DNA Fragments

Contributed by John M. Sedivy, Department of Molecular Biophysics and Biochemistry, Yale University, 333 Cedar Street, New Haven, CT 06510.


Introduction
Genes of higher eukaryotes can be hundreds of kilobases in size. The need to rapidly clone and manipulate DNA molecules in that range has become acute in conjunction with the Human Genome Project. Cosmid vectors are constrained to 4045 kb by the packaging limit of phage lambda. Recently, three systems have been developed that extend this range: yeast artificial chromosome (YAC)-based vectors (>500 kb limit),1 phage P1-based vectors (100 kb limit),2 and E. coli F factor-based vectors (limit as yet untested).35 YAC clones have proven very successful, but are difficult to manipulate due to the extreme fragility of large linear DNA molecules. Bacterial episomes, being supercoiled, dont suffer that constraint. Unfortunately, F factor vectors have been limited by the low efficiency of transformation of large DNA molecules into E. coli. Recently, through the use of electroporation, efficiencies of 1010 transformants/g DNA of small plasmids have been achieved. A systematic analysis of large DNA molecules has been reported,5 and is summarized here.


Materials and Methods
E. coli strain JS46 was grown at 37 C in LB broth; in mid-log phase (OD600=0.6) flasks were placed in ice water and chilled for 15 minutes. Cells were harvested, washed in ice-cold 1 mM HEPES, pH 7.0 (first wash 1x, second wash 0.5x of original culture volume), and resuspended at 1011 cells/ml in 10% glycerol. 40 l aliquots were flash-frozen in dry ice/ethanol and stored at -70 C. Cells were thawed on ice, DNA (2 l in TE) was added, and transferred to ice-cold 0.2 cm gap Bio-Rad electroporation cuvettes. A single pulse was delivered (Gene Pulser II apparatus used with the Pulse Controller II) at 25 F, 2.50 kV and 200 ohms (time constant=4.54.7 msec). Cells were immediately resuspended in 1 ml of SOC (room temperature) and incubated at 37 C for 45 minutes. Dilutions were plated on LB plates containing appropriate antibiotics. Plasmid DNA concentrations were determined by absorbance readings at OD260 and verified by gel electrophoresis.


Results
We have investigated the relationship between plasmid size and electroporation efficiency in E. coli and found that even very large plasmids can be efficiently transfected. Six plasmids were used: pUC19 (2.6 kb),7 cosmid tsA/Y/1d4 (44 kb),8 and F factor clones pMBO52 (52 kb), pMBO74 (74 kb), pMBO99 (99 kb) and pMBO136 (136 kb).3 The average efficiencies (number of colonies per g DNA) were: pUC19, 2 x 109; tsA/Y/1d4, 6.1 x 107; pMBO52, 6.6 x 106; pMBO74, 3.2 x 106; pMBO99, 2.7 x 106; pMBO136, 1.7 x 106 (Figure 1). The data points can be fitted to a straight line when both axes are plotted on a logarithmic scale. The relation can be written as E=1.2 x 106M-0.4248, where E is efficiency (colonies/fmole), and M is plasmid size (kb).

Supercoiled and relaxed episomes were electroporated with equal efficiency. Rearrangements incurred during electroporation were analyzed at the level of restriction digestion; none were detected. To exploit these observations we have constructed a novel mammalian- E. coli shuttle vector, designated pFRS1 (F Replicon Shuttle, Figure 2), which contains three functional units: 1) the E. coli F factor origin of replication, partitioning, and copy number control region, 2) a marker selectable both in E. coli and mammalian cells (the neo cassette from plasmid pRSVneo), and 3) the polylinker from the pBluescript SK cloning vector. The vector stably confers resistance to kanamycin in E. coli, and resistance to G418 in mammalian cells. The plasmid is approximately 12 kb in size, and, like its F factor parent, is maintained at low copy number.

Discussion
The new vector system should accept inserts well in excess of 100 kb. The efficiencies of electroporation are well above the minimum required to construct representative libraries of complex eukaryotic genomes. At this time, the only vector system available for the cloning of very large DNA fragments uses YAC vectors. E. coli has several advantages over yeast as an organism for the passive propagation of foreign DNA. E. coli grows faster, DNA can be rapidly and efficiently extracted, and yields, even of single copy plasmids, are significantly higher. In addition, circular supercoiled DNA molecules are physically quite stable and easy to handle. The most crucial issue, however, is the genetic stability of foreign DNA. Use of E. coli offers the distinct advantage of the very considerable body of knowledge concerning its recombination pathways. In several instances the instability of foreign DNA in E. coli has been overcome by the judicious choice of appropriate, multiply marked, recombination-deficient strains.911


References
1. Burke, D. T., Carle, G. F., and Olson, M. V., Cloning large segments of exogenous DNA into yeast by means of artificial chromosome vectors, Science, 236, 806812 (1987).

2. Sternberg, N., Bacteriophage P1 cloning system for the isolation, amplification, and recovery of DNA fragments as large a 100 kilobase pairs, Proc. Natl. Acad. Sci. USA, 87, 103107 (1990).

3. OConnor, M., Peifer, M., and Bender, W., Construction of large DNA segments in Escherichia coli, Science, 244, 13071312 (1989).

4. Hosoda, F., Nishimura, S., Uchida, H. and Ohki, M., An F factor based cloning system for large DNA fragments, Nucl. Acids Res., 18, 38633869 (1990).

5. Leonardo, E. D. and Sedivy, J. M., A new vector for cloning large eukaryotic DNA segments in Escherichia coli, Bio/Technol., 8, 841844 (1990).

6. Sedivy, J. M., Capone, J. P., RajBhandary, U. L., and Sharp, P. A., An inducible mammalian amber suppressor: propagation of a poliovirus mutant, Cell, 50, 379389 (1987).

7. Yanisch-Perron, C., Vieira, J., and Messing, J., Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors, Gene, 33, 103119 (1985).

8. Schnipper, L. E., Chan, V., Sedivy, J. M., Jat, P. S., and Sharp, P. A., Gene activation by induced DNA rearrangements, Cancer Res., 49, 66406644 (1989).

9. Wyman, A. R., Wolfe, L. B., and Botstein, D., Propagation of some human DNA sequences in bacteriophage 1 vectors requires mutant Escherichia coli hosts, Proc. Natl. Acad. Sci. USA, 82, 28802884 (1985).

10. Ishiura, M., Hazumi, N., Koide, T., Uchida, T., and Okada, Y., A recB recC sbcB recJ host preve nts recA-independent deletions in recombinant cosmid DNA propagated in Escherichia coli, J. Bacteriol., 171, 10681074 (1989).

11. Leach, D., and Lindsey, J., In vivo loss of supercoiled DNA carrying a palindromic sequence, Molec. Gen. Genet., 204, 322327 (1986).


pBluescript is a registered trademark of Stratagene Cloning Systems.


back to top
'"/>

Source:


Page: All 1 2 3 4 5

Related biology technology :

1. Efficiently Insert Unique Restriction Sites into Plasmid Vectors
2. Efficient Cleavage of Fusion Proteins to Yield Native Amino Termini
3. Mammalian Expression Vector for Efficient Cloning of PCR Fragments
4. Efficient Transfection of Neurospora Crassa
5. Efficient Transfection of Neurospora Crassa
6. Efficient Recovery of Ultrapure Plasmid DNA
7. Efficient and Reliable PCR Setup Using Eppendorf MasterMix
8. Efficient DNA transfection of primary CNS neurons using TransMessenger Transfection Reagent
9. Efficient RNAi-mediated gene silencing in neuronal cells using QIAGEN siRNA and TransMessenger Transfection Reagent*
10. Cloning Based on Efficient Three-Fragment Assembly DNA Ligation
11. Efficient and Reliable Linear Amplification of cRNA
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:6/23/2016)... 23, 2016  The Biodesign Challenge (BDC), a university ... to harness living systems and biotechnology, announced its winning ... New York City . ... showcased projects at MoMA,s Celeste Bartos Theater during the ... MoMA,s senior curator of architecture and design, and ...
(Date:6/23/2016)... ... 23, 2016 , ... STACS DNA Inc., the sample tracking software company, today ... Laboratory, has joined STACS DNA as a Field Application Specialist. , “I am ... and COO of STACS DNA. “In further expanding our capacity as a scientific integrator, ...
(Date:6/23/2016)... , June 23, 2016  Blueprint Bio, a company ... to the medical community, has closed its Series A ... Nunez . "We have received a commitment ... capital we need to meet our current goals," stated ... us the runway to complete validation on the current ...
(Date:6/23/2016)... ... June 23, 2016 , ... ... the Pennsylvania Convention Center and will showcase its product’s latest features from June ... be presenting a scientific poster on Disrupting Clinical Trials in The Cloud during ...
Breaking Biology Technology:
(Date:4/19/2016)... -- The new GEZE SecuLogic access control ... system solution for all door components. It can be ... interface with integration authorization management system, and thus fulfills ... dimensions of the access control and the optimum integration ... considerable freedom of design with regard to the doors. ...
(Date:4/14/2016)... 2016 BioCatch ™, the ... announced the appointment of Eyal Goldwerger as ... Goldwerger,s leadership appointment comes at a time of ... deployment of its platform at several of the world,s ... discerns unique cognitive and physiological factors, is a winner ...
(Date:3/31/2016)... March 31, 2016  Genomics firm Nabsys has completed ... Barrett Bready , M.D., who returned to the ... original technical leadership team, including Chief Technology Officer, ... Development, Steve Nurnberg and Vice President of Software and ... company. Dr. Bready served as CEO of ...
Breaking Biology News(10 mins):