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June 12-13, 2019 | Edinburgh, Scotland
8th European Clinical Microbiology and Immunology Congress
&
3rd World congress on Biotechnology
Joint Event
Microbiology: Current Research | Volume: 3 | ISSN: 2591-8036
Characterization of recombinant plasmid PEPCK-PIRES2-EGFP and evaluation of
PEPCK promoter specificity in hepatocytes cells culture transfected with liposomes
Lucas-González Amellalli
Instituto Politécnico Nacional (IPN), México
G ene therapy consists of introducing foreign genetic
material, DNA or RNA, into cells with genetic disorder,
in order to create beneficial biological effects. In order
to achieve this effect, it is necessary to introduce the
therapeutic genes through a genetic vehicle. Liposomes
are gene delivery vectors with very low immunogenicity
and toxicity, ease of manufacture, furthermore, they lack
DNA insert size limitation. Cationic liposomes seem to be
the most hopeful, due to his ability to interact with the DNA
and the cell surface. Oncematerial genetic gets inside cell, it
can carry therapeutic genes whose expression is regulated
by tissue and organ-specific promoters. PEPCK promoter
is stably active and specific in hepatocytes, so it could be
adapted to therapeutic interest genes and get into the
body for selective expression in liver cells. In this work, the
genetic construction pPEPCK-IRES2-EGFP was characterized
by enzymatic restriction, PCR and nucleotide sequencing,
confirming the presence of the organ specific promoter
PEPCK. Once the identity of the genetic construct was
confirmed, it was used to transfect the hepatocarcinoma
cell line HepG2, the embryonic kidney cell line Hek293FT as
a control of specificity and a primary culture of hepatocytes,
using cationic liposomes from the DLO and DLE lipids as a
delivery vector. The specificity of expression in liver cells was
evidenced by the green fluorescent protein gene contained
in the recombinant plasmid used. This is intended to create
a powerful tool to address the expression of therapeutic
genes in the treatment of liver diseases, without affecting
other cell types.
e:
Microbiol Curr Res, Volume 3
ISSN: 2591-8036