Penggunaan Fusion

Genes dalam Tranfeksi

Sel

Afifa Khairinnisa
AntiAging dan Aesthetic Medicine
 Tranfeksi tekhnik transfer gen untuk
menintro duksi molekul DNA ke dalam sel
 Keberhasilan transfer gen menggunakan
metode tranfeksi ditentukan faktor :
pemilihan larutan tranfeksi
 Sesuai dengan mempertimbangkan
kesediaan secara komersial, mudah
diaplikasikan, keberhasilan tinggi dan
tidak bersifat toksik
Contoh pengenalan DNA asing pada sel
mamalia
Ada beberapa metode tranfeksi
menggunakan bahan kimia yang sering
dilakukan diantaranya dengan
menggunakan kalsium fospat, sediaan
liposom maupun polimer
 Fusion Gene  process by which the whole or
parts of two distinct genes are fused into a
single gene
 Sel tunggal dinamakan hibridoma yg memiliki
sifat kedua sel
 Penyisipan gen reporter ke sekuen umum yg
mengatur ekspresi gen reporter tersebut
 This phenomenon has been broadly observed
in organisms ranging from higher eukaryotes
to prokaryotes
 Proses
 Dalam proses fusi sel, sel B dan sel T
dijadikan sel sumber gen yang memiliki
sifat yang diinginkan yaitu mampu
memproduksi antibodi
 Sel target digunakan sel myelomayg
mampu membelah diri dengan cepat
 Sel B dan sel T difusi dengan sel mieloma
untuk mempercepat fusi sel digunakan
fusi gen
 Tranfeksi merupakan prosedur yang
paling sering digunakan untuk
menganalisa ekspresi gen mamalia secara
in vivo
 Early studies of gene expression relied on
the creation of clonal cell lines that
extremely time-consuming
 However, it was found that chromosomal
location can have significant effects on
gen expression

Using Fusion genes to Measure

Promotor Activity
 Ekspresi gen : proses penentuan sifat
suatu organisme oleh gen
 Transient assay systems that are based
on the use of fusion gene and asses gene
expression within 48 hrs after introducing
of the DNA
 Fusion Gene  easily assayed protein are
critical in establishing optimal tranfection
condition

Characterization of transfection
efficiency through use of fusion
genes
Overview of genetic reporter
system
 Gen Reporter  gen yang digunakan sbg
indikator untuk membedakan organisme
yang berhasil di transformasi, dapat juga
digunakan sebagai indikator keberhasilan
kloning
 Penggunaan gen reporter untuk sel mamalia
pertama kali digunakan pada tahun 1982
 Digunakan vektor plasmid yang berisi gen
pengkode enzim bakteri B-galaktosidase
untuk mempelajari regulasi gen eukaryot
 Refer to procedures in which the reporter
protein is quantified using either cell or
tissue lysates containing the reporter , or
the culture medium from transfected
cells.

In Vitro Reporter Assays

 This enzyme catalyzes the transfer of
acetyl groups from acetyl-CoA to
Chlorampenicol
 CAT is procaryotic enzyme, there are
minimal competing activities in most
eukaryotic cells, resulting in high signal-
to-background rations in these reporter
assays.

Chlorampenicol Acetyltransferase
(CAT)
 Firefly Luciferase
 B-Galactosidase
 Secreted Alkaline Phosphatase (SEAP)
 Human Growth Hormone (hGH)
 B-Glucuronidase (GUS)
 Green Fluorescent Protein (GFP)
 Firefly Luciferase
 B-Galactosidase
 May be difined as those in which the
reporter protein is detected in either live
cells or tissues, or in cells or tissues that
have been fixed for histochemical staining

In Vivo Reporter Assay

 Human somatic cell gene targeting
 Provides a powerful tool to scientists
studying gene function in cultured human
cells.
 importance, Because it can be used to
either delineate the loss-of-function
phenotype of a gene or correct a mutated
gene back to wild-type

Inaktivasi DNA dalam sel mamalia

 Strategic planning
 Selection of parental cell line
Six different human cell lines have been
successfully used for human somatic cell
gene targeting
Cell line : HCT116, DLD1, HT1080, SW48,
HaCaT, Human fibroblast
 Architecture of promotorless targeting
vector
 Promotorless targeting vectors require
that the selectable marker gene be
inserted in-frame into the gene being
targeted.
 There are several general factors to
consider when planning to construct such
a targeting vector
 Building a targeting vector
 Building promotorless targeting vectors is
a nontrivial exercise in recombinant DNA
technology
 2 factors:
 There is virtually no sequence flexibility in
the juction between the left arm of the
targeting vector and the selectable
marker cassette
 Second, it is frequently necessary to add
several sequence features between the
end of the selectable marker gene and the
right homology arm and targeting vector,
 Such as retriction sites and a PCR priming
site
 The simple approach for identifying gene-
targeted cell line is to conduct an initial-
PRC- based screen
 Then confirm the putative knockouts with
a Southern blot

Screening for Knockouts in human

cell