1. In Vivo Gene Modification by CRISPR/Cas-9
System
Cloning the T-vector makes it possible to begin
mutagenesis. Sequencing results demonstrated
that our cells have the sequence of what Figure 5.2
will be. Mutagenesis has been started using the
sequenced results. The sequence that we currently
have will be used as a template to generate Figures
5(a) and 5(d). The PNPLA3 sequence for Figure
5(c) will be generated from Figure 5(a). Once the
four combinations are generated, HepG2 cells will
be transfected to create 4 different cell plates under
the same conditions - in vitro NAFLD – as shown in
Figure 5. Calculating exact fat values is still being
considered so results from the cell cultures will first
be only qualitative.
Background
Objective
Methods
Results
Discussion
Future Direction
Acknowledgements
Non Alcoholic Fatty Liver Disease (NAFLD) is when
“excess fat accumulates in the liver of a patient
without the history of alcohol abuse.” Although exact
causes are unknown, obesity, rapid weight loss,
insulin resistance (diabetes), and genetic factors are
correlated to the disease. Once a person develops
NAFLD, if left untreated, it can progress into
Steatosis, Steatohepatitis, Fibrosis, Cirrhosis, and
even death.
Determine the effect of different combinations
of these SNPS on an ethnic group not known
to be affected by these SNPs
Figure 1 shows the sequencing results of the
genomic DNA of HepG2 cells. Figure 2 is the result
of mRNA from HepG2 cells. In both the genomic
DNA and mRNA results, Figures 1(a) and 2(a),
Methionine was present at the 148th codon instead
of Isoleucine. SNP 1 exists in the HepG2 cells.
Figures 1(b) and 2(b), genomic DNA and mRNA
respectively, had the same results, SNP2 was not
present at the 453rd codon. Theoretically, having
SNP 1 but not SNP2 should make a person much
more susceptible to NAFLD.
Dr. Qi-Long Ying, PI
Chang Liu, Mentor
Ying Lab Members
Dr. Roberta Diaz Brinton, Program Director
Christina Zeitountsyan, Program Coordinator
Mrs. Ramirez – De La Cruz, Program Teacher
CIRM and STAR Program
The patatin like phospholipase domain containing
3 (PNPLA3) gene encodes a lipase that mediates
hydrolysis in adipocytes. If the protein is not
translated, the breakdown of fat is disrupted in the
liver, resulting in fat accumulation and the liver
damage without any alcohol abuse.
In 2009, PNPLA3 gene was identified as significant
to certain ethnic groups’ susceptibility to NAFLD.
The I148M variant, a single nucleotide
polymorphism (SNP), was found to increase
Hispanics‘ susceptibility to the disease. The
second SNP, the S453I variant, was found to make
African Americans less susceptible to the disease.
The HepG2 cells that will be used in this project
are an immortalized cell line that come from a
young, Caucasian male. Because it is unknown
what the effect of the variants are in other ethnic
groups, this is significant.
Hypothesis
Fig. 1 Sequencing results of genomic DNA from HepG2 cells
(a) SNP is present at the 148th codon resulting in change from Isoleucine to Methionine.
The red square indicates a mismatch with the sequence of the program.
(b) SNP not present at the 453rd codon so Serine remains the same
Isoleucine
&
Serine
No SNP
(Wild Type)
Methionine
&
Isoleucine
SNP 1&2
Isoleucine
&
Isoleucine
SNP 2
Methionine
&
Serine
SNP 1
Of the four combinations of SNP’s the one with
SNP 1 but without SNP 2 should retain the
most fat.
Figures 5 (a)-(d) demonstrate the different combinations of SNPs.
(a) (b) (c) (d)
(b)(a)
Fig. 2 Sequencing results of mRNA from HepG2 cells
(a) SNP is present at the 148th codon resulting in change from Isoleucine to Methionine.
(b) SNP not present at the 453rd codon so Serine remains the same
(a) (b)
1. Extract genomic DNA from HepG2 cells and determine sequence at
148th and 453rd codon
2. Extract mRNA from HepG2 cells and generate variants of PNPLA3
HepG2 cells
Extract
genomic
DNA
Send for
sequencing
Extract mRNA
PCR to get
cDNA from
mRNA
T-vector
cloning
vector
insert
Mutagenesis