JoVE Journal

Biology

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Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

16.5K Views

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09:02 min

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January 8th, 2015

DOI :

10.3791/52155-v

January 8th, 2015

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Thimma R. Reddy¹, Emma J. Kelsall¹, Léna M.S. Fevat², Sarah E. Munson*³, Shaun M. Cowley*¹

¹Department of Biochemistry, University of Leicester, ²Center for Fisheries, Environment and Aquaculture Sciences, ³ES Cell Facility, Centre for Core Biotechnology Services, University of Leicester

Summary

Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles. Here, we describe an improved method of recombineering in E. coli, that we term ‘subcloning plus insertion’, which can be used to generate custom gene targeting vectors rapidly.

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Keywords Subcloning

Gene Targeting Vector

Homologous Recombination

Bacterial Artificial Chromosome

Conditional Gene Knockout

Knock in Vector

BAC Reporter Vector

Selection Marker

Multiplex Approach

Plasmid Gap Repair

Chapters in this video

0:05

Title

1:04

Multiplex Recombineering Oligos

2:00

BAC Clone Transformation

3:56

Insertion Cassettes and Subcloning Plasmids

5:03

Subcloning Plus Insertion

7:07

Analysis of Recombinants

7:48

Results: Subcloning Plus Insertion (SPI)

8:39

Conclusion

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