Gene transfer in animals

Gene transfer, methods of gene transfer, Viral and non Viral methods and application

1. GENE TRANSFER TECHNOLOGY IN ANIMALS VIRAL AND NON-VIRAL METHOD PRESENTED BY, RAHUL KUMAR GOSWAMI

2. INTRODUCTION Gene transfer is to transfer a gene and DNA segments from one organism to another. This area of genetic manipulation makes important contributions to domesticated animals in relation to immunology, vaccines, aging, and cancer. Gene transfer represents a relatively new possibility for the treatment of rare genetic disorders and common multifactorial diseases by changing the expression of a person's genes.

3. Gene transfer method in animals There are four major strategies for gene transfer to animal cells, two of which are considered :- biological mechanisms using virus (transduction) Virus ( transduction ); the transferred gene represents part of the viral genome. or bacterial that invade animal cells ( bactofection ) Bacteria ( bactofection ); the gene will be transferred as a plasmid. Biochemical and physical methods which do not involve infection thus termed transfection .

4. The gene transfer results can be transient and stable transfection . Transient transfection In transient transfection , the transfected DNA is not integrated into host chromosome. DNA is transferred into a recipient cell in order to obtain a temporary but high level of expression of the target gene. Stable transfection Stable transfection is also called permanent transfection . By the stable transfection , the transferred DNA is integrated (inserted) into chromosomal DNA and the genetics of recipient cells is permanent changed.

5. Non viral methods (Bio chemical and Physical methods) Biochemical methods Calcium phosphate method; involves the formation of a fine DNA/calcium phosphate co-precipitate which first settles on the cells and then internalized by endocytosis . The precipitate must be formed freshly at the time of transfection . The DNA escapes and reaches the nucleus and can be then expressed. Since the cells must be coated by the calcium complex, monolayers of cells must be used for maximum efficiency. However, this method gives only 1-2% transfection efficiency.

6. DEAE- dextran mediated ( diethylaminoethyloethyl-dextran ) There are three points that DEAE- dextran mediated transfection differs from calcium phosphate coprecipitation . It is used for transient transfection . (2) It works more efficiently with cell lines of BSC-1, CV-1 and COS, etc. (3) It is more sensitive. protocols :- (1) Harvest exponentially growing cells by trypsinization and transfer then into 60-mm tissue culture dished at a density of 105 cells/dish. (2) Add 5 ml complete growth medium. (3) Incubate 24 hours at 37 o C with 5% CO 2 . (4) Prepare DNA/DEAE- dextran /TBS-D solution by mixing 2 mg of superoiled plasmid DNA into 1 mg/ml DEAE- dextran in TBS-D. (5) Remove medium and wash tree times with PBS and twice with TBS-D.

7. DEAE- dextran mediated cont. (6) Add DNA/DEAE- dextran /TBS-D solution 250 ml. (7) Incubate 60 min at 37 o C with 5% CO 2 . (8) Remove DNA/DEAE- dextran /TBS-D solution. (9) Wash with TBS-D three time and PBS twice. (10) Add 5 ml medium supplemented with serum and chloroquine (0.1 mM ). (11) Incubate 4 hours at 37 o C with 5% CO 2 . (12) Remove medium. (13)Wash with serum-free medium three times. (14) Add to cells 5 ml of medium supplement with serum, and incubate 48 hours at 37 o C with 5% CO 2 . (15) Harvest the cells after the 48 hours transfer- ction . (16) Analyze RNA or DNA by hybridization, or analyze expressed protein by radiommunoassay , immunoblotting , immuniprecipitation , or by enxzy moatic activity in cell extract.

8. Lipid mediated method This method can be used for both transient and stable transfection , and it can be used for adherent cells, primary cell lines, and suspension cultures. For the following protocol, the Lipofectamine Reagent from Invitrogen Corporation will be used. Lipofectamine Reagent is a 3:1 (w/w) L iposome formulation of the plycationic lipid 2,3-dioleyloxy-N-[ 2( sperminecardox-ido )ethyl ]-N,N-dimethyl-1-propanaminium trifluoro -acetate (DOSPA) and the neutral lipid dioleoyl phosphatidylethanolamine (DOPE) in membrane-filtered water.

9. Lipid mediated method protocol :- 1) Put about 40,000 cells per well of a 24-well plate in 0.5 ml of the appropriate complete growth medium (add 10% serum if it needs). (2) Incubate the cells at 37 o C in a CO 2 incubator until the cells are 50-80% confluent (about 20 hours, depending on the cells). (3) Dilute 3 mg DNA into 25 ml medium without serum for each well and mix. (4) Dilute 3 ml Lipofectamine Reagent into 25 ml medium without serum for each well and mix. (5) Combine diluted DNA (Step 3) and Lipofect -amine Reagent (Step 4) and incubate at room temperature for 30 min. In this step the DNA-liposome complexes are formed. (6) Replace the medium in the cells with 0.2 ml transfection medium without serum.

10. Lipid mediated method cont. (7) Add 0.15 ml medium without serum to the tube containing the complexes for each well. (8) Incubate the cells with the complexes for about 10 hours at 37 o C in a CO 2 incubator. The incubating time will be flexible by the cell type. (9) Add 0.4 ml growth medium containing double the 2× normal concentration of the serum without removing the transfection mixture. (10) Replace the medium with fresh, complete medium at 20 hours following the start of transfection if continued cell growth is required. (11) Assay cell extracts for transient gene expression at 24-72 hours after transfection , depending on the cell type and promoter activity. (12) To obtain stable transfectants , passage the cells 1:10 into the selective medium after 72 hours of transfection for the reporter gene transfected .  

11. Physical method of gene transfer or transfection in animal (Non viral methods) In this method, naked DNA is deposited directly into the cell by exploiting a physical force. This includes Microinjection particle bombardment ultrasound electroporation Which ever method used, the result is called transformation which is a change in the recipient cell’s genome caused by the acquired transgene .

12. Microinjection Direct transfer of DNA into the cell without a carrier is called DNA microinjectin . This can only be done for only few cells at a time. This technique is used mainly for large cells such as oocytes , eggs and the cells of early embryos. The DNA can be directly injected into tissues, such as skin, muscle or internal organs or it can be injected into the blood.

13. Particle bombardment or Gene gun ( Biolistics ) Some cells, tissues and intracellular organelles are impermeable to foreign DNA To resolve this problem in gene transfer, the gene gun was made by Klein at Cornell University in 1987. The gene gun is part of the gene transfer method called the biolistic (also known as biobalistic or particle bombardment) method. In this method, DNA or RNA adhere to biological inert particles (such as gold or tungsten). By this method, DNA-particle complex is put on the top location of target tissue in a vacuum condition and accelerated by powerful shot to the tissue, then DNA will be effectively introduce into the target cells. Uncoated metal particles could also be shot through a solution containing DNA surrounding the cell thus picking up the genetic material and proceeding into the living cells. The efficiency of the gene gun transfer could be depended on the following factors: cell type, cell growth condition, culture medium, gene gun ammunition type, gene gun settings and the experimental experiences, etc.

14. Particle bombardment or Gene gun ( Biolistics ) cont.

15. Ultrasound Involves the exposure of cells to a rapidly oscillating probe such as the tip of sonicator . In this method the application of ultrasound waves to a dish or cells or a particular tissue results in the formation and collapse of bubbles in the liquid, including the cell membrane, a process known as cavitation . The transient appearance of such cavities allows the DNA to cross the membrane into the cytoplasm. This method can be used both for in vivo or in vitro as the plasmid DNA is left structurally intact.

16. Electroporation is a physical transfection technique involves creating transient nano -meter size pores in the cell membrane by exposing cells to a brief pulse of electricity. The most critical parameter is the intensity and duration of electrical pulse. Steps of the electroporation transfection : (1) Harvest cells in the mid- to late-logarithmic phase of growth. (2) Centrifuge at 500 g (2000 rpm) for 5 min at 4 o C. (3) Resuspend cells in growth medium at concen - tration of 1 X 10 7 cells/ml. (4) Add 20 mg plasmid DNA in 40 ml cells. (5) Electric transfect by 2.1 kV / 25u F for 2.6 milliseconds. (6) Transfer the electroporated cells to culture dish and culture the cells. (7) Assay DNA, RNA or protein and continuously culture the cells to get positive cell lines.

17. Electroporation cont.

18. Biological mechanisms virus mediated gene transfer in animals Virus particles have a natural ability to adsorb to the surface of the cells and gain entry. This can be exploited to deliver recombinant DNA into animal cells. Several classes of viruses has been used for gene transfer and at least 8 has been used in clinical trials. Transgenes may be incorporated into viral vectors either by addition to the whole genome or by replacing one or more viral genes. This can be done by ligation or by homologous recombination. If the transgene replaces a none essential gene the vector is described as helper-independent If it replaces an indispensable gene, then this vector will be helper dependent . It is generally recommended to use vectors from which all viral coding sequences has been deleted such vectors are described as fully deleted or gutted or gutless vectors. These vectors contain just the cis -acting elements required for packaging and for genome replication. High capacity for foreign DNAbecause no viral gene products are made, the vector has no intrinsic cytotoxic effects.

19. virus mediated gene transfer in animals cont. Adeno virus These are viruses with a linear, double stranded genome, of approximately 36 kb. These are used frequently due to certain advantages; Stability high capacity for foreign DNA wide host range including none-dividing cells Ability to produce high titer stocks up to 10 11 pfu /ml

20. Virus mediated gene transfer cont.

21. Adeno -associate virus (AAV) These viruses are not genetically related to adenovirus but is so-called because it was first discovered as a contaminant in an adenovirus isolate. The AAV is a single stranded DNA and is a member of the parvovirus family. It is naturally replicating deficient, thus it requires the presence of another virus such as adenovirus to complete its infection cycle. AAV replicates lytically and produces thousands of progeny virions . The dependence of AAV on a heterologous helper virus such as adeno virus provides an unusual degree of control over vector replication making AAV one of the safest vectors to use for gene therapy. Other advantages of this viral vector is the wide host range that it exhibits including none dividing cells.

22. Adeno -associate virus (AAV) cont.

23. Method The AAV genome is small (5 kb) and comprises a central region containing rep ( replicase ) and cap ( capsid ) genes flanked by 145 kb inverted terminal repeats. Foreign DNA replaces the cap region and gets expressed by indigenous promoter. However rep proteins might interfere with the expression process thus responsible for some of the cytotoxic effects of the virus. Then virus is bind to host cell and use host cell machinary and divide their viral genome. And the viral genome codes the protein.

24. Baculovirus Baculovirus promote high level of transgene expression in insect cells but can also infect mammalian cells. Have rode shape capsid and large double-stranded DNA genomes. They productively infect arthropods particularly insects. BV vectors are used mainly for high level transient protein expression in insects and insect cells.

25. Retrovirus-Mediated Gene Transfer The most useful vectors for the purpose of gene isolation are those that lend themselves to the production of libraries consisting of overlapping fragments of genomic DNA, ideally encompassing the entire genome several times. This can best be done at 4-16 cell stage embryos. However, it can be done up to midgestation , but with incomplete infections i.e low infectivity rate. Immediately following infection, the retrovirus produces a DNA copy of its RNA genome using its reverse transcriptase. Completion of this process requires that the host cell undergoes the S phase of the cell cycle. Therefore, retroviruses effectively transduce only mitotically active cells. Modifications to the retrovirus frequently consist of removal of structural genes, such as gag, pol , and env , which support viral particle formation.

26. Retrovirus-Mediated Gene Transfer cont. Additionally, most retroviruses and complementary lines are ecotropic in that they infect only rodents, such as rats and mice, and rodent cell lines rather than humans. The DNA copy of the viral genome, or provirus, integrates randomly into the host cell genome, usually without deletions or rearrangements. Because integration is not by way of homologous recombination, this method is not used effectively for site-directed mutagenesis. Very high rates of gene transfer are achieved with the use of retroviruses.

27. Retrovirus-Mediated Gene Transfer cont.