University of Science and Technology of Hanoi

Department of Life Science

Major: PMAB
REPORT

Apply crispr-cas9 to treat

Osteosarcoma cells in vitro to open a
new therapeutic strategy for the
disease in Canis familiaris

Member of group:

Lê Mai Chi ( BA10-006)

Nguyễn Hải Hoàng (BI11-098)

Lưu Mộc Linh Hương ( BI11-109)

Trần Ngọc Lân ( BI11-139)

Doãn Nhật Linh ( BI11-143)

Tô Minh Nhật (BI11-208)

1
Table of Contents
ABSTRACT ..................................................................................................................... 3

I. INTRODUCTION: ....................................................................................................... 3

1.1. Canine Model ......................................................................................................... 3

1.2. Epidemiology and Genetic factors of OSA ............................................................... 4

1.3. Introduction to CRISPR-Cas9 ................................................................................ 4

1.4. The TP53 gene in Canis familiaris ........................................................................... 6

2.1. Tissue isolation and preservation: ........................................................................... 8

2.2. Cell culture............................................................................................................. 8

2.3. Guide RNA design .................................................................................................. 9

2.4. Vector construction ................................................................................................ 9

2.5. Canine TP53 target sequencing ............................................................................. 10

2.6. Introduce the vector into the secondary cell lines: ................................................. 10

2.7. MTT assay for A and B dishes: ............................................................................. 11

2.8. Annexin V Staining Protocol for Flow Cytometry: ................................................ 12

III. RESULTS AND DISCUSSION................................................................................. 13

3.1. Results analysis: .................................................................................................. 13

3.2. Positive results: .................................................................................................... 13

3.3. Negative results: .................................................................................................. 14

3.4. Solutions for negative results: .............................................................................. 14

IV. REFERENCES ........................................................................................................ 14

2
ABSTRACT
Nowadays, with the development of people both physically and mentally, dogs have
become one of the spiritual values in the lives of many individuals. Humans live
with them and give them affection. However, no one wants their friend to suffer
from a painful illness. Canis familiaris, especially huge and enormous breeds, are
prone to bone cancer. Osteosarcomas make up 85% of all canine bone tumors.
Osteosarcomas are cancerous tumors that cause painful bone deterioration in the area
where they grow. The most common site of osteosarcoma in dogs is the limbs, but
it can also affect the skull, ribs, vertebrae, and pelvis.It occurs more frequently in
large dogs than in smaller breeds. It occurs more frequently in large dogs than in
smaller breeds. The malignancy will progress to the lungs in approximately 80% of
individuals. Bone cancers' biological behavior, prognosis, and treatment are all
affected by the tumor type, initial location, and extent of disease dissemination. To
establish the most appropriate treatment, a variety of diagnostic tests such as X-rays,
blood tests, and occasionally a biopsy are required.

In humans, bone cancer has been studied by many scientists, but not much has been
done in dogs. For this study, we will focus on the TP53 gene. We apply CRISPR-
Cas9 on the cancer cells then observe whether they can reactivate the TP53 gene’s
function (turn it into the prior tumor suppressor gene with normal function).

I. INTRODUCTION:
1.1. Canine Model
Domesticated dogs are widely acknowledged as suitable models for a variety of
biomedical research fields, as they offer a number of advantages over other often
used experimental animals. The fact that dogs live with their owners, are kept until
death, and generally receive excellent care, including highly-trained medical
attention, is arguably the most intriguing of these. This model of the human state
enables researchers to examine complex issues such as environmental variables in
disease, aging and its impact on disease susceptibility and development, and the
effects of long-term therapy protocols. [1-3]

3
New research indicates that larger breeds have a higher risk of osteosarcoma than
smaller breeds, and that dogs with shorter skulls and legs had a lower risk. The
findings could inform future breeder health adjustments and study into the
development of malignancies tumors in normal bone. [4].

We selected to employ primary cell culture for our sample since these are cancer
cells, which will result in a continuous cell line. Next, we apply the CRISPR CAS-
9 genome editing technology to a cell line containing osteosarcoma from a Boxer
dog which has osteosarcoma in the appendicular bone. Using single strand
conformation polymorphism (SSCP) analysis, the presence of identical mutations in
exons was determined for each type. [4]

1.2. Epidemiology and Genetic factors of OSA

Osteosarcoma is the most prevalent type of primary bone malignancy in dogs. It is

considered that the incidence of OS in dogs is at least 13.9/100,000 [1]. OS is a
malignant bone tumor that causes lameness or pain in dogs and is typically
accompanied by a bony or soft tissue mass or edema [5]. OS develops from
malignant osteoid-producing mesenchymal bone-forming cells [6]. The median age
at which dogs get OS is between middle and old age (7 years) [7]. However, greater
weight (> 25 kg) and a tall shoulder height (90 percent of all OS cases) appear to be
the most significant predictors of the development of this cancer in dogs [8]. Gender
does not influence the likelihood of developing OS [9].

Research demonstrating the presence of the insulin-like growth factor-1 (IGF-1)

receptor and ligand in canine osteosarcoma (OS) cells implies a connection between
canine OS and bone growth [10].

1.3. Introduction to CRISPR-Cas9

Genome editing is a sort of genetic engineering that involves inserting, removing, or
changing DNA in living cells. CRISPR (Clustered Regularly Interspaced Short
Palindromic Repeat) is the name given to the unusual organization of short, partially
repeated DNA sequences present in prokaryotes' genomes. CRISPR and its related
protein (CAS-9) are a type of adaptive immunity used by prokaryotes to protect
themselves from viruses and bacteriophages. [11]

4
1.3.1. CRISPR-Cas9 Components
A "guide" RNA (gRNA) and a non-specific CRISPR-associated endonuclease
(Cas9) or other orthologue are the two components of CRISPR. The gRNA is a short
synthetic RNA with a Cas9-binding "scaffold" sequence and a tailored 20-nucleotide
"spacer" or "targeting" motif that defines the genomic target to be changed. Cas9's
genomic target can thus be changed simply by modifying the targeting sequence in
the gRNA.
RuvC and HNH, two functional endonuclease domains in the Cas9 nuclease, cleave
opposing strands of target DNA [12].

1.3.2. CRISPR-Cas9 Mechanisms

Gene knockouts, gene activation, repression, epigenetic gene editing (methylation,

demethylation), and single-base nucleotide modifications (see picture above) are all
possible using the CRISPR/Cas9 system.

The presence of Protospacer Adjacent Motif (PAM) downstream and upstream from
the target is required for target specificity between the gRNA and the complementary
locus. The PAM sequence is required for target binding, and the exact sequence
varies for every Cas9 species. The Cas9 protein and the gRNA produce a riboprotein
complex after they are both expressed. The Cas9-gRNA combination will bind to
any genomic sequence with a PAM, but whether Cas9 will cleave DNA is
determined by how closely the gRNA spacer matches the target DNA. [12]

5
The ability to use the CRISPR/Cas system as a genome editing approach in
eukaryotes, on the other hand, stems from the cell's unique repair mechanisms when
a double strand break occurs in the DNA double helix.

In this case, the eukaryotic cell uses one of two DNA-repair mechanisms: the error-
prone Non-Homologous End Joining (NHEJ), which requires direct ligation of
broken DNA ends, or the error-free Homology-Dependent Repair (HDR) process.
[13]

Because the NHEJ is an error-prone mechanism, the repairs are usually imperfect
due to nucleotide insertions, deletions, or substitutions occurring at the site of
breakage, which frequently results in the gene's "loss of function." The HDR repair
mechanism, on the other hand, requires the presence of a repair template DNA
molecule, such as a sister chromatid, and has been used to perform precise gene
modifications such as coding sequence replacements, targeted mutagenesis, gene
correction, and targeted transgene insertion [14].

1.3.3. Why do we use CRISPR-CAS9

The CRISPR-Cas9 gene-editing technique has revolutionized transgenic animal

production. This technology has demonstrated unequaled efficacy, multiplex
capability, and usability, reducing the time and cost of genome editing and enabling
the production of animals with more extensive genetic modifications. It has also
been demonstrated to be effective in a vast array of animals, from early-branching
metazoans through primates. Consequently, this provides us the notion to adapt this
method to our research on dogs.

1.4. The TP53 gene in Canis familiaris

The TP53 gene controls the synthesis of p53 tumor protein (or p53). This protein
controls cell division by stopping cells from expanding and dividing (proliferating)
too rapidly or uncontrolled.

The p53 protein is located in the nucleus of cells throughout the entire body, where
it binds to DNA. When the DNA of a cell is damaged by agents such as harmful
chemicals, radiation, or ultraviolet (UV) rays from the sun, this protein is crucial in
determining whether the DNA is repaired or the injured cell self-destructs (undergo
6
apoptosis). If DNA damage is fixable, p53 activates other genes to fix it. If DNA
damage cannot be repaired, this protein stops cell division and initiates apoptosis.
p53 contributes to the prevention of tumor formation by stopping the division of
cells with mutant or damaged DNA. Because p53 is crucial for DNA repair and cell
division regulation, it has been called the "guardian of the genome." [15].

Loss of normal function of the p53 tumor suppressor gene product is one of the most
prevalent changes in a wide range of human malignancies, as well as several animal
cancers. The majority of p53 mutations in human cancers are found in four of the 53
protein's critical, highly conserved domains, which are located in exons 5-8. The
homologue of p53 in dogs has been identified, and mutations in this gene have been
seen in thyroid and mammary cancers in this species. In terms of histology,
biological behavior, and response to cytostatic treatment, spontaneously occurring
osteosarcomas in dogs and humans share many similarities. The location of the
appendicular skeleton is the most common. Axial skeleton tumors in dogs are less
common than appendicular skeleton tumors, and they appear to have a lower rate of
metastasis, but a high rate of local recurrence. [16]

The TP53 gene encodes the P53 protein. It works as a tumor suppressor in a variety
of tumor forms, inducing growth arrest or apoptosis depending on the physiological
conditions and cell type. As a trans-activator involved in cell cycle regulation, it
inhibits cell division by regulating a collection of genes necessary for this process.
One of the active genes is an inhibitor of cyclin-dependent kinase. It appears that the
induction of apoptosis is mediated by either the upregulation of BAX and FAS
antigen expression or the downregulation of Bcl-2 expression. [17]

Through the Rb pathway, p53 inhibits cell cycle progression. Activated p53
stimulates the transcription of mdm2, a negative regulator of p53 protein stability,
and cki p21, which inhibits Rb phosphorylation, in response to cellular stress and/or
DNA damage.

In response to DNA damage, hypoxia, or chemotherapeutic drugs, tumor cells

carrying active oncogenes also experience p53-mediated apoptosis. [23]

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II. MATERIALS AND METHODS
2.1. Tissue isolation and preservation:
The SSCP method was applied to evaluate a dog with osteosarcoma of the
appendicular bone. We carried out surgical procedures on the canine in order to
remove tumor tissue that was malignant. After the removal of fragments of tissue
from the organ in a sterile environment, the cell suspension that is obtained is washed
with a physiological buffer.

[16]

2.2. Cell culture

2.2.1. Making Culture Medium grow cells isolated from Osteosarcoma tumor
tissue (“OSCA” medium)
1. Sterilize hood and set up a 250ml bottle with proper fitting Bottle top Filter (keep
vacuum off until step 3).
2. Prepare OSCA Media (DMEM media supplemented with 10% FBS) by
combining the below into a bottle top filter:
● 222ml DMEM-with high glucose [GIBCO 11965]
● 25ml FBS (10%)
● 0.5ml Primocin [Invivogen, # ant-pm-1]
● 2.5ml HEPES [Cellgro, #25-060-C1]
● 250ml Total

3. Turn Vacuum on and Filter the media through.

[18]
2.2.2. Obtain the primary cell culture
1. Pour 5ml OSCA media to a petri dish.
2. Spread out the cell suspension to the surface of the petri dish we were prepared.
The appendicular OS cells are put into the dish.
3. Put the dish into the CO2 incubator (37°C, 5% CO2) to provide the appropriate
environment for cell growth.

8
 4. Leave the newly cultured cells for 2-3 days.
[19]
2.2.3. Obtain the secondary cell culture to conduct CRISPR-Cas9:
1. Take the dish, remove the spent medium and wash in PBS to remove dead cells
and serum.
2. Add 2 ml trypsin and incubate at 37°C, 5% CO2 for 3-5 minutes
3. Prepare 2 petri dishes, pour 5ml OSCA media in each dish.
4. Transfer the cells from the dish into two new dishes. Sign as dish A and B.

2.3. Guide RNA design

To create gRNAs targeting the canine TP53 locus, all candidates containing NGG-
protospacer adjacent motif (PAM) sequences in all exons of the gene were
discovered using the Target finder tool developed by the Feng Zhang laboratory
[20].

Since the program's automatic off-target screening technique was unavailable for
dog species, we restricted the findings using the steps below. First, gRNAs close to
the N-terminus within the third exon of each gene were chosen because their
functional domains may create partially functional products. Using the Basic Local
Alignment Search Tool (BLAST), candidates with a decreased binding potential
relative to other genomic regions (gRNAs max score 30.2), particularly to coding
sequences, were selected to avoid off-target modifications. Nineteen gRNAs for
TP53 were selected using this process and the final two gRNAs were selected based
on their lowest non-specific binding potential, in accordance with the Max score
determined using the BLAST algorithm. [21]

2.4. Vector construction

For the construction of a CRISPR/Cas9 plasmid targeting canine TP53, the U6-
stuffer-hSpCas9 sequence was digested with Kpn1 (Klenow fragment) and BamH1
from lentiCRISPRv2. This segment was inserted into the Nru1 and BamH1 sites of
the pcDNA3.1(+) genome (Thermo Fisher Scientific, Waltham, MA, USA). The
enhanced green fluorescence protein (EGFP) sequence from pEGFP-N2 (Clontech
Laboratories Inc., Mountain View, CA, USA) was digested with EcoR1 and Not1

9
and the E2A peptide sequence was introduced into the pcDNA3.1-CRISPR plasmid.
The three gRNA sequences were then generated and put into the two BsmB1 sites
flanking the stuffer sequence. [21]

2.5. Canine TP53 target sequencing

Using a Wizard Genomic DNA Extraction Kit (Promega, Madison, WI, USA),
genomic DNA was isolated from canine fetal fibroblasts in accordance with the
manufacturer's instructions. Exon 6 of TP53 was amplified in each cell line using
Takara Ex taqTM (Takara Bio Inc., Shiga, Japan) and primer sets forward 5′-CCT
CAA CCG CCT CAC AAA-3′ and reverse 5′-ATT CCT CCC CGA TGG CTC
TTA-3′. Each product was ligated according to the manufacturer's recommendations
into a pGEM T-easy vector (Promega) and then sequenced using an ABI BigDye®
terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA)

[21]

2.6. Introduce the vector into the secondary cell lines

1. Transfer the cells in the dish into 2 tubes, sign as tube 1 and tube 2.
2. Add the CRISPR/Cas9 components into each tube.
3. Put the tube into the electroporator to induce the formation of temporary pores
in the plasma membrane and the electrical potential across the membrane causes
charged molecules (e.g., gRNA/Cas9) to enter the cytoplasm through the pores.
4. After that, transfer the cells from each tube into the petri dishes, still signed as A
and B.
5. We have 2 dishes:
● A: Contains CRISPR-Cas9 introduced appendicular bone osteosarcoma
cells.
● B: Contains wild-type appendicular osteosarcoma cells. (Control group)
After 2 or 3 days, continue to subculture them continuously.
6. Days by days, conduct observations on each dish.

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2.7. MTT assay for A and B dishes
- MTT Solution:
1. Dissolve MTT in Dulbecco’s Phosphate Buffered Saline, pH=7.4 (DPBS) to
5 mg/ml
2. Filter-sterilize the MTT solution through a 0.2 µM filter into a sterile, light
protected container.
3. Store the MTT solution, protected from light, at 4°C for frequent use or at -
20°C for long term storage.

- Solubilization Solution:
1. Choose an appropriate solvent resistant container and work in a ventilated
fume hood.
2. Prepare 40% (vol/vol) dimethylformamide (DMF) in 2% (vol/vol) glacial
acetic acid.
3. Add 16% (wt/vol) sodium dodecyl sulfate (SDS) and dissolve.
4. Adjust to pH = 4.7
5. Store at room temperature to avoid precipitation of SDS. If a precipitate forms,
warm to 37°C and mix to solubilize SDS.
- MTT Assay Protocol:
1. Prepare cells in each dish and test compounds in 96-well plates containing a
final volume of 100 µl/well.
2. Incubate for desired period of exposure.
3. Add 10 µl MTT Solution per well to achieve a final concentration of 0.45
mg/ml.
4. Incubate 1 to 4 hours at 37°C.
5. Add 100 µl Solubilization solution to each well to dissolve formazan crystals.
6. Mix to ensure complete solubilization.
7. Record absorbance at 570 nm.

[24]

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2.8. Annexin V Staining Protocol for Flow Cytometry
1. Prepare:
• 12 x 75 mm round-bottom tubes.
• 1X PBS .
• Annexin V Apoptosis Detection kit (any one of the kits listed). Each kit
includes an Annexin V conjugate.
° eFluor 450 (Cat. Nos. 88-8006-72, 88-8006-74)
° FITC (Cat. Nos. 88-8005-72, 88-8005-74)
° PerCP-eFluor 710 (Cat. Nos. 88-8008-72, 88-8008-74)
° PE (Cat. Nos. 88-8102-72, 88-8102-74)
° PE-Cyanine7 (Cat. Nos. 88-8103-72, 88-8103-74)
° APC (Cat. Nos. 88-8007-72, 88-8007-74)
• 10X Binding buffer.
2. Prepare 1X binding buffer by mixing 1 part of 10X binding buffer with 9
parts of distilled water.
3. Harvest cells.
4. Wash cells once in 1X PBS, then once in 1X binding buffer.
5. Resuspend cells in 1X Binding Buffer at 1-5 x 106 cells/mL.
6. Add 5 μL of fluorochrome-conjugated Annexin V to 100 μL of the cell
suspension.
7. Incubate 10-15 minutes at room temperature. Protect from light.
8. Add 2 mL 1X binding buffer and centrifuge at 400-600 x g for 5 minutes at
room temperature. Discard supernatants.
9. Resuspend cells in 200 µL of 1X binding buffer.
10. Add 5 μL of Propidium Iodide Staining Solution or 7-AAD Viability
Staining Solution and incubate 5-15 minutes on ice or at room temperature.
Note: Propidium iodide and 7-AAD must remain in the buffer during
acquisition. Do not wash cells after the addition of propidium iodide or 7-AAD.
11. Analyze by flow cytometry.
Note: Cells should be analyzed within 4 hours after the initial incubation period
due to adverse effects on the viability of cells left in the presence of propidium
iodide or 7-AAD for prolonged periods. Store at 2–8°C and protect from light
until ready for analysis.[25]

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III. RESULTS AND DISCUSSION
3.1. Results analysis
- CRISPR-Cas9 will reactivate the mutant tp53 gene, hence causing osteosarcoma
cells to undergo apoptosis.
- Based on the findings of the MTT test, the percentage of live cells in the devices
was computed and compared to the percentage of live cells in dishes A and B.
- Active live cells generate dehydrogenases that reduce MTT, resulting in the
formation of violet formazan crystals. The amount of formazan crystals produced
in the test will reflect the percentage of cells in which the Tp53 gene has been
awakened.
- If the function of the Tp53 gene has been reactivated, the cells will undergo
apoptosis and become dead; consequently, there will be less purple formazan
crystals. In conclusion, the less number of crystals, the more efficient the
CRISPR-Cas9 technique against the OS cells.
- The number of formazan crystals formed was assessed by OD optical
densitometry at 570 nm.
- Next, we have to undergo the Flow cytometry - one of the most popular and
versatile applications for studying whether the cells undergo apoptosis or just
necrosis. In our technique, Annexin V binding to phosphatidylserine residues
normally located within the plasma membrane. Phosphatidylserine residues are
externalized during apoptosis, so only cells that have decided to die will be
detected by annexin V binding.
[26]
3.2. Positive results
- If our technique is effective, the MTT assay will reveal that the quantity of
formazan crystals in dish A is substantially smaller than in dish B => CRISPR-
Cas9 is effective at reviving mutant Tp53, hence inducing cancer cell death.
- Next, the flow cytometry findings demonstrate that in dish A, cells die due to
apoptosis rather than necrosis, as proof that there were no manipulation errors in
the laboratory activity.

13
3.3. Negative results
- If our approach is ineffective, the MTT assay will show that the amount of
formazan crystals in dish A is similar to that in dish B, indicating that CRISPR-
Cas9 is ineffectual at reviving mutant Tp53 and hence not driving cancer cell
death.
- In another case, the flow cytometry results show that in dish A, cells die owing
to necrosis rather than apoptosis, indicating that there were manipulation errors in
the laboratory activity.
3.4. Solutions for negative results
- If CRISPR-Cas9 cannot directly reactivate the Tp53 gene, we have to choose an
indirect method. We can produce the chimeric antigen receptors (CARs) by
CRISPR- which are synthetic immunoreceptors recently used in cancer
immunotherapy, serving as regulators of malignant cell adhesion to proteins
presented on the extracellular membrane.

[27]
- However, if we wish to stick with the concept, we can do so in a different exon.
- With manipulation errors, it can be corrected by repeating the experiment until
the desired results are achieved.

IV. REFERENCES
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dog breeds: their impact on canine medicine and the use of the dog as a
preclinical animal model. AAPS J. 10(1):110-119.
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human as companion model systems. Mam. Genome 18(6-7):444-451.
4. Edmunds, G.L., Smalley, M.J., Beck, S. et al. Dog breeds and body
conformations with predisposition to osteosarcoma in the UK: a e-control
study. Canine Genet Epidemiol 8, 2 (2021).

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occurring disease to inform pediatric oncology. ILAR J. 2014;55(1):69–85.
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Osteosarcoma, Cancer Treatment and Research, Vol. 152. New York:
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Clinical Oncology, 5th edition. St. Louis, MI: Saunders Elsevier. p 463–503.
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osteosarcoma. Vet J 156:31–39.
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tumors in dogs: Technique description and outcome in 9 dogs. Vet Surg
34:24–34.
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M, Radinsky R. 2004. IGF-1 receptor contributes to the malignant phenotype
in human and canine osteosarcoma. J Cell Biochem 92:77–91.
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CRISPR/Cas-9-Mediated Genome Editing. Biologics : targets & therapy, 15,
353–361.
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R (2013) One-step generation of mice carrying mutations in multiple genes
by CRISPR/cas-mediated genome engineering. Cell 153:910–918
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Kirpensteijn, G.R. Rutteman. P53 gene mutations in osteosarcomas in the dog,
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19. Srijana Khanal. Animal Cell Culture: Types, Applications. 5/5/2022
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21. Kiyoung Eun, Min Gi Park, Yeon Woo Jeong, Yeon Ik Jeong, Sang-Hwan
Hyun, Woo Suk Hwang,Sung-Hak Kim and Hyunggee Kim. Establishment of
TP53-knockout canine cells using optimized CRISPR/Cas9 vector system for
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Minor. Cell Viability Assays. May 1, 2013
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Cytometry
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Therapy and Beyond. Trends Mol. Med. 2017;23:430–450. doi:
10.1016/j.molmed.2017.03.002

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