Different types of Gene Transfer Methods

1. Gene Transfer Methods

2. Gene
• A gene is the basic unit of heredity in a living
organism
• Genes are working subunit of DNA/RNA

3. Gene Transfer
– The insertion of genetic material into a cell
– It is defined simply as a technique to efficiently
and stably introduce foreign genes into the genome
of target cells
– The insertion of unrelated, therapeutic genetic
information in the form of DNA into target cells
Bacteria (or) Virus
Animal
Plant

5. Applications of Gene Transfer- Bacteria

6. Applications of Gene Transfer- Plants
• Provide resistance against viruses.
• Acquire insecticidal resistance.
• To strengthen the plant to grow against bacterial diseases.
• Develop the plants to grow in draught.
• Engineering plants for nutritional quality.
• Make the plants to grow in various seasons.
• Herbicide resistant plant can be made.
• Resistance against fungal pathogens.
• Engineering of plants for abiotic stress tolerance.
• Delayed ripening can be done.

7. • Clinical gene transfer applications
• Vaccine Development
• Production of transgenic animals
• Treatment of Cancer, AIDS
• Gene Discovery
• Gene Therapy
• GMO
Applications of Gene Transfer- Animals

8. Types of gene transfer
• Natural method
– Conjugation
– Transformation
– Transduction
– Vector mediated
– Transposon
• Artificial method
– Physical method
– Chemical method
– Electrical method

9. Artificial method
• Physical method
– Microinjection
– Macroinjection
– Gene gun (Biolistic)
– Laser transfection
– Sonication

10. Artificial method
• Chemical method
–Calcium Phosphate
–PEG
–Liposome
–DEAE Dextran (Diethylaminoethyl)
• Electrical method
–Electroporation
–Electrofusion

11. Conjugation
• Requires the presence of a special plasmid called
the F plasmid.
• Bacteria that have a F plasmid are referred to as
as F+ or male. Those that do not have an F
plasmid are F- of female.
• The F plasmid consists of 25 genes that mostly
code for production of sex pilli.
• A conjugation event occurs when the male cell
extends his sex pilli and one attaches to the
female.

12. Conjugation
• This attached pilus is a temporary cytoplasmic
bridge through which a replicating F plasmid
is transferred from the male to the female.
• When transfer is complete, the result is two
male cells.
• When the F+ plasmid is integrated within the
bacterial chromosome, the cell is called an Hfr
cell (high frequency of recombination cell).

13. Conjugation

14. Transformation
• Transformation is the direct uptake of exogenous
DNA from its surroundings and taken up through
the cell membrane .
• Transformation occurs naturally in some species
of bacteria, but it can also be effected by artificial
treatment in other species.
• Cells that have undergone this treatment are said
to be competent.
• Any DNA that is not integrated into he
chromosome will be degraded.

15. Transformation

16. Transduction
• Gene transfer from a donor to a recipient by way
of a bacteriophage
• Two way of integration
• If the lysogenic cycle is adopted, the phage
chromosome is integrated (by covalent bonds)
into the bacterial chromosome, where it can
remain dormant for thousands of generation
• The lytic cycle leads to the production of new
phage particles which are released by lysis of the
host.

17. Transduction

18. Vector Mediated Gene Transfer
• Carrying molecule related gene transfer
–Plant vector- CaMV, TMV
•Agrobacterium mediated gene
transfer
–Animal vector- Sv40, Pox Viral vector
–Bacterial vector- pSC 101

19. Agrobacterium Mediated Gene Transfer
• The Agrobacterium system was historically the first
successful plant transformation system, marking the
break through in plant Genetic engineering in 1983.
• Large plasmids (140–235 kbp) in these bacteria are
called tumour inducing (Ti plasmid) and root inducing
(Ri plasmid) respectively.
• Virulence genes aid in the transfer of T-DNA into the
host plant cell. Ti plasmid contains 35 vir genes
arranged in 8 operons.
• Integrated in the plant nuclear DNA at random site

20. Agrobacterium Mediated Gene Transfer
Advantages
• Agrobacterium is capable of infecting infect plant cells,
tissue and organs.
• Capable of transfer of large fragments of DNA very
efficiently
• Integration of T DNA is a relative precise process.
• The stability of gene transferred in excellent.
Limitations :
• Host specificity
• Somaclonal variation
• Slow regeneration
• Inability to transfer multiple genes

22. SV 40
• Simian virus 40 vector belongs to family polyoma
virus family.
• This virus induces tumours to animal cells and used
for gene transfer into eukaryotic cells
• Sv40 diameter is 45nm and its capsid contains 72
proteins
• It doesn’t have enzymes in structure
• Genome of sv40 is single molecule of double
stranded DNA
• It contain 5243bp in genome structure

23. SV 40

24. Transposons
• Transposon, class of genetic elements that can
“jump” to different locations within a genome.
• In addition, most transposons eventually
become inactive and no longer move.

25. Microinjection
•DNA introduced into cells or protoplasts with use of very fine needles or
glass micropipettes
•T. P. Lin in 1966 (mouse eggs)
•The DNA solution is injected directly inside the cell using capillary glass
micropipettes with the help of micromanipulators of a microinjection
assembly.
•It is easier to use protoplast than cells since cell wall interferes with the
process of microinjection.
•The protoplast are usually immobilized in agarose (or) on a glass slides
coated with polylysine or by holding them under suction by a micropipette.
•The process of microinjection is technically demanding and time
consuming a maximum of 40-50 protoplasts can be microinjected in one
hour.
•The transformation frequency ranging between 14 to 66 %.

26. Microinjection

27. Microinjection
•Advantages
– Stable integration better than other method.
–DNA injected in this Process less modification only
occur
–Effective transforming primary cells and protoplast.
•Limitations
–Cost effective
–Random integration
–Only one cell at a time can be manipulated.
–Method not useful for the walled cells
–Skilled person required

28. Macroinjection
•DNA Macroinjection employing needles each with
diameter greater than cell diameter
•De la Pena et al., 1987
•The injection of DNA into the cells or tissues using
hypodermic syringe is known as macro injection.
•It is hypothesized that the DNA is taken up by
microspores during some specific stage of their
development.
•This approach is also very simple and easy; the only
problem concerns the frequency (0.07%) and the
consistency of stable transformants obtained.

29. Macroinjection

30. Macroinjection
•Advantages
–Instrument simple and cheap.
–Does not require Protoplast.
–Technically simple.
•Limitations
–Less specific
–Less efficient
–Frequency of transformation is very low.

31. Gene gun
• Biolistics method/Microprojectile/Particle bombardment
• Sanford and coworkers in 1987 (onion epidermal)
• Klein et al., transferred genomic RNA (TMV)
• The process of transformation employees foreign DNA
coated with (0.2-0.7 μm ) gold (or) are tungsten particles
to deliver into target plant cells.
• Two procedures have been used to this techniques
– i. By using pressurized helium gas.
– ii. By electro static energy released by a droplet of
water exposed to a high voltage.

32. Gene gun

33. Gene gun
• Advantages
– Need of protoplast obtaining can be avoided
– Walled intact cells can be penetrated
– Genome of subcellular organelles can be
manipulated.
• Limitations
– Random injection
– Need of equipment
– Damage cell

34. Laser
• It is method of introducing DNA into plant cells with a
laser micro beam.
• Small pores in the membrane are created by laser micro
beam.
• Lasers puncture holes in the cell membrane through
which DNA may enter into the cell cytoplasm.
• This method also used in Plant cells as well as Animal
cells.
• But, There is no information/report on gene expression
or stable integration.

35. Laser

36. Sonication
• Sonoporation involves the use of ultrasound for temporary
permeabilization of the cell membrane allowing the uptake of
DNA, drugs or other therapeutic compounds from the
extracellular environment.
• This method leaves the compound trapped inside the cell after
ultrasound exposure.
• It employs of micro bubbles for enhancing the delivery of
large molecules like DNA. The micro bubbles form complex
with DNA followed by injection and ultrasound treatment to
deliver DNA into the target cells.
• Unlike other methods of transfection, sonoporation combines
the capability to enhance gene and drug transfer.

37. Sonication

38. Sonication
Advantages
• Simple and highly efficient gene transfer method.
• No significant damage is cause to the target tissue.
Limitations
• Not suitable for tissues with open or complex
structures.
• High exposure to low-frequency (<MHz) ultrasounds
result in complete cellular death (rupture of the cell).
• Thus cellular viability must be taken into
consideration while employing this technique.

39. Calcium Phosphate
• Transfection is most common method
• Graham and Vander E.B. in 1973.
• A fraction of cells will take up the calcium
phosphate DNA precipitate by endocytosis.
• Transfection efficiencies using calcium
phosphate can be quite low, in the range of
1-2 % .
• This technique is used for introducing DNA
into mammalian cells.

40. Calcium Phosphate

41. Advantages
–Mostly in mammalian cells.
–Along with other method
Limitations
–Small fraction of cells is stably integrated
–Frequency very Low
–Gene modification
–1-5% DNA enter into the nucleus.
–Toxic especially to primary cells
Calcium Phosphate

42. • This method is utilized for protoplast only.
• Polyethylene glycol stimulates endocytosis and
therefore DNA uptake occurs.
• Protoplasts are kept in the solution containing
polyethylene glycol (PEG).
• The molecular weight of PEG used is 8000
Dalton having the final concentration of 15%.
• CaCl2 or sucrose or Glucose – osmotic buffer
reagent .
• Mostly plant system (petunia)
PEG

43. • Advantages
–More efficient than electroporation
–Useful for plant
• Limitations
–Transformation not high
–Applicable only protoplast
–Increases nucleases effect.
PEG

44. • Artificial phospholipid vesicles
• Lipofection.
• Fraley in 1988
• DNA complex with liposomes
• Liposome +ve charage
• Membrane – membrane fusion
• Endocytosis
Liposome

45. Liposome

46. • Advantages
–Simplicity
–Long term stability
–Low toxicity
–Protection of nucleic acid from degradation
• Limitations
–DNA passes from cytoplasm to the nucleus
unknown
Liposome

47. • Polycationic high molecular weight
• Endocytosis
• Along with DMSO and PMMA (Polymethyl-
methacrylate) - high
• Viral infection in cell lines.
• 80% transformed can express.
• Transfer efficiency increased by Glycerol.
DEAE Dextran

48. DEAE Dextran

49. • Advantages
–Simple
–Cheap
–Not survive calcium phosphate use this method
• Limitations
–Stable expression is difficult
–Degrade cells
DEAE Dextran

50. • Zimmermann et al., 1983
• Electrical shock (Charge)
• Suspending solution
• Electroinjection
• Types
–High voltage short duration
–Low voltage long duration
Electroporation

51. • Advantages
–Method is applicable to variety of cell lines
–Method quick
–Large number cells handle simultaneously
–Less cost
• Limitations
–Degradation of DNA
–Cell structure
–Clumping of cell
Electroporation

53. • Mostly protoplast
• Mixed fusion
• Two types fusion (Cybrid, Hybrid)
• Along with PEG
• Sensitive to high PEG
Electrofusion

54. Cybrid Formation
Hybrid Formation
Different Cells
Electrofusion

55. • Advantages
–Fusion very easy
–Fuse more cells
• Limitations
–Degradation of DNA
–Cell structure
–Clumping of cell
–Integrated DNA may be modified
Electrofusion