Molecular Biology C
Conception, theory, research, and application
——Logic and LIY (Learn It Yourself)

Sheng Zhao (赵晟), Biochemistry and Molecular Department of Medical school in
Southeast University

Couse QQ Club: 112342994 (分子生物学C )

Web: http://teaching.ewindup.info/

Email: shengzhao@seu.edu.cn or windupzs@gmail.com

QQ /MSN/Skype/gChat: windupzs@gmail.com

Mobile:18551669724 or 13675130010
Chapter 4: Finding Scientific Nemo (From
clinic samples to scientific research)
Section 1:
Trust it or trash it?
——Number, selection, and target (Establish sample
library)
Section 2:
A colorful microscopic world
——Specific labeling (Visualization techniques)
Case 4:
Immortal clinic samples
——Primary culture and gene manipulation (Construction of
the cell line)
HeLa
---- An immortal cell line
吕一正
A HeLa cell, also Hela or hela cell, is
a cell type in an immortal cell line used in
scientific research.
It is the oldest and most commonly used
human cell line.
Henrietta Lacks
(August 1, 1920 – October 4, 1951)
On January 29, 1951, she felt a knot inside her.
She had told her cousins about the knot; they
assumed correctly that she was pregnant. But,
after giving birth to her fifth child, Joseph,
Henrietta started bleeding abnormally and
profusely. Her local doctor tested her for syphilis,
which came back negative, and referred her to
Johns Hopkins.
Howard Jones, her new doctor, examined
Henrietta and the lump in her cervix. He cut off
a small part of the tumor and sent it to the
pathology lab. Soon after, Lacks learned she
had a malignant epidermoid carcinoma of the
cervix.
Two samples of Henrietta's cervix were
removed—a healthy part and a cancerous
part—without her permission.The cells from her
cervix were given to Dr. George Otto Gey.
Multiphoton fluorescence image of cultured HeLa cells with a fluorescent protein
targeted to the Golgi apparatus (orange), microtubules (green) and counterstained
for DNA (cyan).
Multiphoton fluorescence image of HeLa cells stained with
the actin binding toxin phalloidin (red), microtubules (cyan)
and cell nuclei (blue).
Scanning electron micrograph of an apoptotic HeLa cell.
HeLa cells dividing under electron microscopy
Scanning electron micrograph of just-divided HeLa cells.
Chromosome number
• Horizontal gene transfer from human papillomavirus 18
(HPV18) to human cervical cells created the HeLa
genome which is different from Henrietta Lacks' genome
in various ways, including its number of chromosomes.
HeLa cells are rapidly dividing cancer cells, and the
number of chromosomes varied during cancer formation
and cell culture. The current estimate (excluding very
tiny fragments) is a "hypertriploid chromosome number
(3n+)" which means 76 to 80 total chromosomes (rather
than the normal diploid number of 46) with 22-25 clonally
abnormal chromosomes, known as HeLa signature
chromosomes.The signature chromosomes can be
derived from multiple original chromosomes making
challenging summary counts based on original
numbering. Researchers have also noted how stable
these aberrant karyotypes can be.
Telomerase
• The HeLa cell line was derived for use in cancer
research. These cells proliferate abnormally rapidly,
even compared to other cancer cells. Like many other
cancer cells, HeLa cells have an active version
of telomerase during cell division, which prevents the
incremental shortening of telomeres that is implicated
in aging and eventual cell death. In this way the cells
circumvent the Hayflick Limit, which is the limited
number of cell divisions that most normal cells can
later undergo before becoming senescent.
Contamination
• The degree of HeLa cell contamination among
other cell types is unknown because few
researchers test the identity or purity of alreadyestablished cell lines.
• It has been demonstrated that a substantial
fraction of in vitro cell lines — estimates range
from 10% to 20% — are contaminated with
HeLa cells. Stanley Gartler in 1967 and Walter
Nelson-Rees in 1975 were the first to publish on
the contamination of various cell lines by HeLa.
In vivo magnetic resonance imaging
tracking of bone marrow-derived
mesenchymal stem cells via
intracoronary administration: Consistency
to pathohistological results
李浩
133772
Recent trials and clinical
studies have shown that
intracoronary transplantation
of bone marrow-derived
mesenchymal stem cells
(MSCs) improves cardiac
function following acute
myocardial infarction (AMI).
However, whether homing of
MSCs into the infarcted
myocardium or not is still
unknown.
Epicardial myocardium injection
Endocardial myocardium injection
Intracoronary injection
Intravenous injection
Methods
Methods
Myocardial infarction was induced in all 10 pigs
Methods
Making cell lines and gene deliver system

Oncogenes including myc, bcl-xl, neu, p53, Ela, SV40, among which mcy is the
most used.

Deliver system:
1.
Physical method: Electroporation, Nano particles
2.
Chemical system: Bipolar molecules
3.
Biologic system:

Virus: wide spectrum, smaller inserts
1.

Retroviral (genomic integration)

Retrovirus (RV, replicative, ~10 kb genome)

Lentivirus (LV, both replicative and non-replicative, ~10 kb genome)
2.
Adenovirus (AD, epichromosomal, both replicative and non-replicative, ~10
kb genome)
3.
Adeno-associated virus (AAV, 4.3 kb genome)
Bacteria: Larger inserts, limited hosts cells
1.
Salmonella sp
2.
Clostridium sp
3.
Escherichia coli
comparing and optimizing approaches
133763
133764
蔡蓉蓉
胡姗姗
Objectives

 Investigate the feasibility of achieving high transgene
expression in cultured muscle cells.
Methods

materials
mouse
C2C12 cells
a novel system,
primary rainbow
trout myosatellite
cells
Methods

three different viral
vectors
adeno-associated
virus
baculovirus (BAC)
serotype 2 (AAV2)
green fluorescent protein (GFP)
lipofectamine
2000
lentivirus
the genetic manipulation of
cultured muscle cells

1. chemical/lipid-based transfection:
Chemical transfections are fast and cost effective
in most cell culture systems, but produce low transfection
efficiencies in muscle cells.
2.viral vectors:
capable of introducing transgenes in a tissue-specific manner
and may or may not integrate transgenes into the host genome,
depending on the specific vector
The poor transfection efficiency
of lipid-based protocols

• GFP expression was lost as cells proliferated and
was poorly retained after differentiation.
• The reagent was toxic at very low cell densities.
• In general, transfection efficiencies with cationic
lipids are inversely related to transgene size.
Adeno-associated viral
vectors

Adeno-associated viral vectors have great promise for gene
therapy as they infect both dividing and nondividing cells,
they rarely integrate into host genomes and they are
relatively immune tolerant.
Serotype:AAV2、AAV6、AAV8
AAV2

•In this study, we used AAV2 due to the comparatively
lower transduction efficiency of AAV6 in vitro
•In fact, a preliminary trial in our laboratory using both
vector systems clearly demonstrated AAV2 superiority,
even when vector was maintained on cells for 6 day
•Disadvantage：The transgene expression did not continue
after cells were passed
BAC

•BAC can transduce a broad range of mammalian cells as
well and has emerged as a promising gene delivery vector
due to low cytotoxicity and the ability to carry large inserts
•It also transduced myoblasts and in a shorter time than
AAV2, although a 10-fold higher titer was needed to
produce the same relative transduction efficiency.
Promoters

 CMV :The cmv activity is more variable in different cell types
in vitro
 PGK: The pgk promoter was most effective in C2C12 cells
 although cmv and pgk appeared equally efficient in trout
myosatellites, promoter activity of both was comparatively
weak and could explain the need to use a 10-fold higher MOI
to detect significant fluorescence.
Lentiviral

 Lentiviral superiority over the other vectors is likely due to
the fact that their genomes, the vector provirus, are integrated
into host genomes.
 Our results also demonstrate that lentivirus itself does not
alter the differentiation potential of C2C12 myoblasts.
 lentivirus effectively and rapidly infects myoblasts, at lower
virus titers, and that transgene expression is maintained well
after differentiation
Can the nervous system
regenerate ?

The amount of neurons remains unchanged and the
nervous system loses the ability of proliferation and
differentiation since one person is born?

何骏驰
• The immortalization of wild type hNSC
with v-myc results in the establishment of
a stable neural stem cell line (v-IhNSC)
with the ability to originate mature
functional neurons and conspicuous
amounts of oligodendrocytes in vitro.
• 1. Propagation and differentiation of neural stem
cells
• Cells were derived from brain of a aborted
human fetus, cultured in the serum-free medium
containing all needed nutrition, and were
induced to differentiation.
• 2. Retroviral infections
• Retroviral vector encoding v-myc was used to
transduce hNSCs at 17 passages from the
primary culture. A mock vector was used as a
control. Retroviral transduction was a repeated
infection procedure. Then v-IhNSCs were
induced to differentiation.