Bài giảng Nhập môn công nghệ sinh học - Transfection

us-based gene transfer system consists of two components: 
- the transfer vector , which harbours a foreign gene 
linked to elements needed for retroviral replication 
- the packaging cell , which supplies the necessary 
retroviral proteins 
Viral vectors mechanism to deliver their nucleic acid 
- By its stable integration into a chromosome, 
genetic material introduced by a retroviral 
mechanism will remain in the body forever 
unless the targeted cells and their progeny are 
eliminated. 
- This advantage concerns only divinding cells. 
- Recently, the use of HIV-1 and other 
lentiviruses showed that these vectors may 
enter also nondividing cells 
TRANSFECTION : BIOLOGICAL METHODS 
2.2. Adenoviruses 
Adenovirus vectors have proven able to mediate 
long lasting expression in nondividing or slowly 
dividing cells. 
TRANSFECTION : different approches 
E x vivo : 
- A sample of the patient's cells can be removed and 
exposed to the vector in a laboratory setting. The cells 
containing the vector are then returned to the patient 
In situ : 
- The vector can be injected directly into a specific 
tissue in the body, for example : tumor 
In vivo : 
- The vector can be injected intravenously (by IV) 
TWO TYPES OF TRANSFECTIONS 
1. Transient transfection 
When the introduced DNA remains in the cell for a short time, independent of the chromosome. 
2. Stable Transfection 
For many experiments, short-term studies using transiently transfected plasmids are not 
sufficient. It is often necessary to have the gene of interest integrated into the host chromosomes 
for long-term analysis . 
- Some vectors are used for transient transfection , others for stable transfection 
Procedure : 
To obtain stable transfection , we perform in well 
plates according to the protocol for transfection in 
adherent cells. Then, we start selection with 
appropriate antibiotic 24–48 h after transfection . 
The selectable marker is often a gene conferring 
antibiotic resistance. 
Cells expressing the gene can survive in antibioticcontaining 
medium, while cells without the 
integrated DNA will die. 
DNA CLONING 
What is DNA cloning ? 
 Restriction enzymes 
 DNA ligases 
 Types of vectors : plasmids 
 Reporter genes 
 Isolation of plasmid from E. Coli 
 Electrophoresis 
 Dosage of DNA 
DNA CLONING 
Whay is DNA Cloning ? 
DNA cloning is the art of creating recombinant DNA molecules that can be : 
- introduced into living cells 
- replicated 
- passed on the daughter cells as that cell divides 
DNA cloning has been made possible by the discovery of two types of 
protein : 
- Restriction enzymes which break such that they have suitable termini 
for ligation 
- DNA ligases which are capable of ligating the molecules of DNA 
DNA to be inserted 
Restriction enzymes 
DNA ligases 
Recombinant plasmid 
DNA CLONING 
1. Restriction enzymes or endonucleases : 
- Discovered in the late 1960. 
- Recognize specific DNA sequences and make double-strand cleavages. 
- Most restriction enzymes typically recognize DNA sites that are 4–8 base pairs in length. 
- Bacterial species contain restriction–modification systems with genes that encode both a restriction endonuclease and a methyltransferase that recognizes the same sequence. The host DNA is fully protected from the action of the restriction enzyme by the methyltransferase 
DNA CLONING 
2. DNA Ligases 
- DNA strands can be joined through the action of the enzyme DNA ligase . 
- The normal biological role of the ligase , is to join the series of Okazaki fragments 
during replication. 
- « T4 DNA Ligase » is used in cloning experiment (E. Coli) 
DNA ligases are able to connect between 3'- 
OH carbone and phosphate-5' of two 
neighboring nucleotides in the DNA strand . 
DNA CLONING 
3. 1. Types of vectors : Plasmids 
- Plasmids are the most widely used vectors. 
- It is very easy to isolate them from the host cell. 
- They are extrachromosomal genetic elements which control their own replication. 
- Bacterial plasmids are circular and double-stranded that range in size from 1 to 10 kb. 
The fact that bacterial plasmids are nonessential for their host organism, 
and have a size and form suitable for manipulation, has made possible 
their widespread applicability to DNA cloning 
DNA CLONING 
3.1. Types of vectors : Plasmids 
Promoter : to initiate high levels of transcription (T7) 
Origin of replication : sequence reconigzed by host cell DNA replication systems. 
X R Selection : gene that encode resistance to antibiotic 
MCS : multicloning site, recognition sites for restriction enzymes (region where forein DNA is to be inserted 
DNA CLONING 
Aschematic representation of a typical cloning experiment. 
(a) The vector is cut within its multicloning site (MCS). 
(b) The target DNA is cut so as to produce termini compatible with the vector. ©The vector and insert are ligated to produce recombinant DNA. 
(d, e) Recombinant DNA is introduced into appropriate host cells. In this illustration the vector encodes resistance to an antibiotic, X. 
(f) If the cells are plated out on to medium containing X, only cells that have been transformed will grow and divide to form colonies 
Isolation of Plasmid DNA from E. coli 
Essentially all genes can be expressed by using recombinant methods. Gene expression in Escherichia coli is the most efficient system. 
1. Transformation 
- After the transformation, the bacteria are cultured in growth medium. 
Since a plasmid usually harbours a selectable marker encoding an antibiotic overcoming protein, the corresponding antibiotic is added to the growth medium to maintain the plasmid in the growing bacterial cells. Coincident with bacterial growth, the plasmid DNAs are also replicated 
Isolation of Plasmid DNA from E. coli 
2. Principle to purify plasmid : 
- Lysis of bacterial cells on modified SDS-alkaline 
- Selective binding of the DNA to silica beads 
- The yield of plasmid is up to and for midi-prep 100 μg and 500 μg maxi-prep 
- The protocols provide a simple and reliable way for rapid isolation of plasmid. 
- The high-quality plasmid DNA is eluted and recovered by ethanol precipitation. 
Plasmid thus prepared can be used immediately for many plasmid routine applications such as 
- DNA sequencing 
- restriction digestion 
- in vitro transcription 
- library screening 
- ligation 
- transformation . 
Microbiology laboratory 
- Incubator (37°C) 
- Temperature controlled Shaker 
- Refrigerated centrifuge 
- Electroporator 
- Hood 
- Ice 
- Refrigirator 
DNA CLONING : Electrophoresis 
Why electrophoresis ? 
- To verify if the purified plasmid obtained after the transformation is correct. 
- Plasmid is digested using restriction enzyme 
Principle of Gel Electrophoresis 
- Electrophoresis is the migration of charged particles or molecules in an electric field. This 
occurs when the substances are in aqueous solution. 
- The speed of migration is dependent on the applied electric field strength and the charges of 
the molecules. 
Agarose gels are used as thick layers in flatbed chambers mainly for preparative purposes. 
DNA CLONING : Electrophoresis 
Electrophoresis setup 
The bands are visualized with 
fluorescent dyes that are visible 
in UV light using ethidium 
bromide. 
Migration of deoxyribonucleic acid fragments 
The relative mobilities of deoxyribonucleic acid 
(DNA) molecules are dependent on the sizes of 
the molecules. 
DNA CLONING : Dosage of DNA 
MOLECULAR BIOLOGY LABORATORY 
- PCR System 
- Centrifuge 
- Hood 
- Spectrophotometer 
- Electrophoresis apparatus 
- Generator with cuve 
- UV transilluminator 
UV transilluminator 
APPLICATIONS 
Transgenic models 
 Gene therapy 
 Antisense strategy 
APPLICATIONS 
Gene transfer technology in vitro is used for many purposes : 
1. To produce large amounts of proteins 
2. To evaluate the effects of specific mutations introduced in genes 
3. To verify the identity of a cloned gene 
4. To study the physiological consequences of overexpression of a 
protein of interest 
APPLICATIONS 
2. Transgenic models : 
Transgenic mice expressing Lac Z 
Transgenic mice expressing 
Growth hormon 
APPLICATIONS 
Transgenic mice expressing GFP 
In transgenic animals, expression can be 
monitored when a given tissue is subjected 
to certain promoter-responsive stimuli 
APPLICATIONS : Gene Therapy 
- Gene therapy is designed to introduce genetic material into cells to compensate for abnormal genes or to make a beneficial protein . 
- If a mutated gene causes a necessary protein to be faulty or missing, gene therapy may be able to introduce a normal copy of the gene to restore the function of the protein . 
- Certain viruses are often used as vectors because they can deliver the new gene by infecting the cell. The viruses are modified so they can't cause disease when used in people. 
- Some types of virus, such as retroviruses, integrate their genetic material (including the new gene) into a chromosome in the human cell. Other viruses, such as adenoviruses, introduce their DNA into the nucleus of the cell, but the DNA is not integrated into a chromosome 
APPLICATIONS : Clinical trials 
TYPES OF VECTORS	 PROTOCOLS 
Retroviruses 	50% 
Adenoviruses	 15% 
Chemical vectors and DNA nude 	20% 
Others	 15% 
APPLICATIONS : Clinical trials 
Approaches to Gene Therapy for Cancer 
There are different approaches to gene therapy: 
- Augmentation of immunotherapy 
- Gene replacement therapy 
- Drug sensitization 
- Drug resistance 
APPLICATIONS : Antisense strategy 
Antisense Nucleic Acids in Biotechnology 
- The strategy of antisense nucleic acid technology is to inhibit expression of a particular gene in a cell. 
- This is achieved by the base pairing of a complementary 
( antisense ) oligo - or polynucleotide to a target RNA, which is usually a messenger RNA (mRNA).