TY - JOUR
T1 - Genetic transformation of BCG
AU - Lugosi, L.
AU - Jacobs, W. R.
AU - Bloom, B. R.
N1 - Funding Information:
Valuable advice and discussion with Scott Snapper and Raul Barletta are gratefully acknowledged. The research was supported by Grant Al23545 from the NIH, a’grant from the Rockefeller Foundation, and grants from the WHO Special Programmes for Research and Training in Tropical Diseases (IMMLEP) and for Vaccine Development (IMMTUB).
PY - 1989/9
Y1 - 1989/9
N2 - Two substrains of BCG, the Pasteur and Japanese, were successfully transformed with E. coli-mycobacteria shuttle plasmids, constructed from the E. coli plasmid, pIJ666 and the M. fortuitum plasmid, pAL5000. Individual plasmids (pYUB13, pYUB14) were obtained that contain selectable antibiotic resistance markers for kanamycin and chloramphenicol resistance that can replicate in both E. coli and BCG. Transformation of two substrains of BCG was successfully accomplished in 8 14 experiments by means of electroporation, and assessed by the growth of kanamycin-resistant colonies. The E. coli plasmid pIJ666 alone was unable to effect transformation. The results suggest that the M. fortuitum sequences required for transformation function as an origin of replication in BCG. The introduction, persistence and the identity of the plasmids were monitored by re-isolation from consecutive subcultures and restriction analysis. The variables associated with transformation, including the age, viability, and glycine pretreatment of BCG cultures, as well as the electroporation parameters on transformation frequencies are analysed. Consecutive transformations of BCG with plasmid DNA isolated from a BCG transformant increased the efficiency from the level of 101-102 obtained with the initial library to 103-104 colonies/μg DNA with functional pYUB plasmids. The hybrid plasmids were genetically stable and maintained expression of kanamycin resistance in continuous subcultures containing kanamycin for 250 generations. The introduction and stable expression of foreign DNA in BCG on a plasmid vector establishes a basis for the construction of polyvalent recombinant BCG vaccine vehicles expressing not only putative protective mycobacterial antigens, but also antigens for other infectious and malignant diseases.
AB - Two substrains of BCG, the Pasteur and Japanese, were successfully transformed with E. coli-mycobacteria shuttle plasmids, constructed from the E. coli plasmid, pIJ666 and the M. fortuitum plasmid, pAL5000. Individual plasmids (pYUB13, pYUB14) were obtained that contain selectable antibiotic resistance markers for kanamycin and chloramphenicol resistance that can replicate in both E. coli and BCG. Transformation of two substrains of BCG was successfully accomplished in 8 14 experiments by means of electroporation, and assessed by the growth of kanamycin-resistant colonies. The E. coli plasmid pIJ666 alone was unable to effect transformation. The results suggest that the M. fortuitum sequences required for transformation function as an origin of replication in BCG. The introduction, persistence and the identity of the plasmids were monitored by re-isolation from consecutive subcultures and restriction analysis. The variables associated with transformation, including the age, viability, and glycine pretreatment of BCG cultures, as well as the electroporation parameters on transformation frequencies are analysed. Consecutive transformations of BCG with plasmid DNA isolated from a BCG transformant increased the efficiency from the level of 101-102 obtained with the initial library to 103-104 colonies/μg DNA with functional pYUB plasmids. The hybrid plasmids were genetically stable and maintained expression of kanamycin resistance in continuous subcultures containing kanamycin for 250 generations. The introduction and stable expression of foreign DNA in BCG on a plasmid vector establishes a basis for the construction of polyvalent recombinant BCG vaccine vehicles expressing not only putative protective mycobacterial antigens, but also antigens for other infectious and malignant diseases.
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U2 - 10.1016/0041-3879(89)90046-9
DO - 10.1016/0041-3879(89)90046-9
M3 - Article
C2 - 2694552
AN - SCOPUS:0024349907
SN - 0041-3879
VL - 70
SP - 159
EP - 170
JO - Tubercle
JF - Tubercle
IS - 3
ER -