Phage particle-mediated gene transfer of recombinant cosmids to cultured mammalian cells.
An efficient procedure for the introduction of recombinant cosmids into cultured mammalian cells consists of the following steps. Cosmids were packaged, in vitro, into lambda phage particles and transduced into Escherichia coli hosts lysogenized with thermo-inducible lambda c Its phage. The introduced cosmids were repackaged into phage particles in the thermo-induced hosts. The efficiency of such in vivo cosmid packaging was further improved by construction of pTC vectors that carried three cohesive end sites (cos) of phage lambda, arrayed in tandem. Two types of cosmids, in almost equal numbers (i.e., cosmids with one cos and cosmids with two cos), were obtained from a cosmid library constructed with pTC vectors. The efficiency of packaging in vivo of cosmids with two cos, was found to be 7-20 times higher than that of corresponding cosmids with only one cos. Use of a high-copy-number derivative of pTCl further improved the phage yield by 20- to 30-fold. The packaged cosmids, which carried the thymidine kinase-encoding gene of herpes simplex virus type 1 as a selective marker, were introduced into mouse Ltk- cells with an efficiency of 10(-5), by the phage transfer method [Ishiura et al., Mol. Cell. Biol. 2 (1982) 607-616].
Ishiura
M
M
National Institute for Basic Biology, Aichi, Japan.
Ohashi
H
H
Uchida
T
T
Okada
Y
Y
D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D003360:Cosmids; D007739:L Cells; D051379:Mice; D011994:Recombinant Proteins / Q000235:genetics; D013937:Thymidine Kinase / Q000235:genetics
D011994:Recombinant Proteins; D013937:Thymidine Kinase