Retrofitting high molecular weight DNA cloned in P1: introduction of reporter genes, markers selectable in mammalian cells and generation of nested deletions.
The bacteriophage P.1. cloning system is proving to be quite useful for the cloning and analysis of genomic DNA inserts of up to 95 kb in size. In an effort to use that DNA directly in biological experiments we have embarked on a scheme to retrofit the P.1. DNA using a mini-Tn10 transposon system. This transposon system is used in two ways: (i) to introduce a variety of sequence signals that are recognizable in mammalian cells, such as mammalian cell-responsive resistance markers and reporter genes, and (ii) to generate a nested set of deletions in a P.1. clone by using a ioxP site located within the transposon. In this report we show that such transpositions into P.1. DNA are efficient, distributed throughout the entire length of the genomic fragment and do not disrupt the DNA in any location other than the site of insertion of the transposon. The Tn10-based P.1. transduction system described here provides a general scheme for retrofitting any large genomic DNA cloned in a P.1. vector, thus facilitating the use of clones from the current P.1. recombinant libraries in cellular transformation studies.
Chatterjee
P K
PK
DuPont Merck Pharmaceutical Company, Glenolden, PA 19036, USA. chatterp@vax.cs.hscsyr.edu
Sternberg
N L
NL
D000818:Animals; D017101:Bacteriophage P1 / Q000235:genetics*; D001665:Binding Sites; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000737:chemistry / Q000235:genetics; D004251:DNA Transposable Elements; D017930:Genes, Reporter; D005819:Genetic Markers; D005822:Genetic Vectors; D019426:Integrases / Q000235:genetics / Q000378:metabolism; D008322:Mammals; D008970:Molecular Weight; D011995:Recombination, Genetic; D017384:Sequence Deletion; D014764:Viral Proteins
D004251:DNA Transposable Elements; D005819:Genetic Markers; D014764:Viral Proteins; D004247:DNA; C045073:Cre recombinase; D019426:Integrases