Method of gene transfer

1. Transfection and Method of
Gene transfer
Dr.Sumedha S.Bobade
Ph.D Scholar
Animal Biotechnology

2. Transfection
• A heritable change in the genome of host by introduction of
the foreign gene manually which express a new phenotype is
known as Transfection.
• DNA which is used for transfection may be cDNA ,genomic
DNA ,genomic DNA clones and retroviral vector.

3. Methods of Transfection
There are two methods of transfection
• Vector mediated
1.Bacteriophage vector
2.Retroviral vector
3.Cosmid vector
4.Baculovirus vector
5.Plasmid vector
• Non vector mediated
There are two main types
1.Direct method
a) Microinjection
b) Embryonic stem cell-mediated gene
transfer.
c) Eloctroporation
d) Particle Bombardment gun.
e) Laser Method
2.Indirect Method
a)Calcium phosphate DNA co precipitation
b) DEAE –dextran mediated

4. Vector mediated Method of
Transfection
Retrovirus vector: RNA viruses which replicate via the
synthesis of a DNA provirus, which is integrated into the
chromosomal DNA infected cell.
A DNA copy of viral RNA is synthesize by enzyme reverse
transcriptase.
This can be modified from original retroviruses that can be
propagated in bacteria as plasmid ,have foreign DNA ligated
into them and transfected into cells.

5. Retrovirus-mediated gene transfer
• For any of these techniques the success rate in terms of live birth of animals
containing the transgene is extremely low.
• Depending on the technique used, the F1 generation may result in
chimeras. When the transgene has integrated into the germ cells, the so-
called germ line chimeras are then inbred for 10 to 20 generations until
homozygous transgenic animals are obtained and the transgene is present in
every cell. At this stage embryos carrying the transgene can be frozen and
stored for subsequent implantation.

6. Somatic cell nuclear transfer
• In 1997, a sheep clone was produced by nuclear transfer (NT ) of a somatic
mammary gland cell into an oocyte.
• The nucleus of the somatic cell is then injected into the enucleated
oocyte, which is transplanted into female recipients.Fibroblast cells
are typically used for NT .
• The adverse effects of the NT technique include a low in utero embryo
survival rate and poor health of the newborn animals.

7. Embryonic stem cell-mediated
gene transfer.
• This method involves prior insertion of the desired DNA sequence by
homologous recombination into an in vitro culture of embryonic stem (ES)
cells.
• Stem cells are undifferentiated cells that have the potential to differentiate
into any type of cell (somatic and germ cells) and therefore to give rise to a
complete organism.
• These cells are then incorporated into an embryo at the blastocyst stage of
development. The result is a chimeric animal.

9. Microinjection
• Lin (1960) first time describe the technique of microinjection of mouse egg .
• It is a technique of delivering foreign DNA into a living
cell/egg/oocyte/embryo of animal through microinjection .
• For microinjection micropipette is used.
• Process of microinjection is done under stereomicroscope .
• Holding pipette holds a target cell to be microinjected at its tip when the cell
gently sucked.
• The tip of micropipette is injected through the membrane of cell.
• The content of needle are delivered into cytoplasm and it is then taken out.
• The injected DNA integrates randomly with nuclear DNA and its expression
could be possible only when the foreign DNA is attached to a suitable
prometer sequence.
Injecting micropipette
Fig :Microinjection technique
Egg Holding pipette

11. Advantages and Disadvantages
• Advantages:
1.This method can not applied for large number of cells.
2.This is useful method for producing transgenic animals.
Disadvantage:
1.Only a very small amount (one picolitre) of transgene is inserted.
2.There is no control over place of insertion of transgene .Frequency of
transgene is very low.
3.There is no control over the insertion of copies of transgene i.e. how many
copies have been inserted.Integrated gene undergo sustantial modification
i.e. recombination, rearrangement and deletion.Integration with host
genome is random.
4.Pronucleus in the fertilized oocyte is difficult to visualize.
Mice and rabbit nucleus is easily easily visualize while pronucleus in sheep
,goat,pig,cattle, and buffaloes are difficult to visualize by ordinarary
method for this contrast microscopy is used.

12. Electroporation
• Electroporation was first describe by Sendai and his coworkers and latter it
was modified by Zimmermann Schewich.
• Electroporation is defined as a physical process that transiently
permeablized cell (both prokaryotic and eukaryotic) plasma membrane with
an electric pulse ,permitting cell uptake of a variety of biological
molecules (Chang,1989).
• The electric field is used to transfer foreign DNA into a fragile cell.
• The short, high voltage DC electric field pulses applied to eukaryotic cell
plasma membranes cause the destablization of the phospholipid layer
which result in the formation of temporary pores in the plasma membrane
,thus allowing an exchange of extracellular and intracellular
macromolecules.

13. Electroporation
• Electroporation instrument consist of
• i) Special chamber having suspended cell with two different commonly
used electropulse electrode :Gene pulser and ECM 830
• ii) An effective buffer which provides optimized condition for
transfecting primary and hard to transfect cell.
• iii) The pulse generator
• iv) An oscillator to moniter the wave form and amplitude of generated
pulse.

15. Advantages and Disadvantages
• Advantages:
• 1) Electroporation has been preffered over PEG as the apparatus deliver
DNA effectively at appropriate electric pulse.
• 2.This process is cost effective and treatment is both reproducible and
simple to apply.
• 3)It has proved to be broadely applicable gene transfer technique,
facilitating foreign gene expression in protoplast as well as intact cell
• 4.Nacked DNA may be used for gene therapy by applying electroporation
devicein animal cell.
• 5.It provides a valuable alteration to chemical and physical method ,that
may be ineffective or toxic when transforming certain cell type.

16. Disadvantage
• 1. As PEG fusion this technique rely on protoplast for introduction of
foreign gene.
• 2.Treating eukaryotic cell with colemid before electroporation increse
transformation efficiency because drug arrest cells in metaphase the cell
lack a nuclear membrane or have unusually permeable membrane.

17. Laser Method
• A brief pulse focuses laser beam used for the transfection was described
by Kurata et al., 1986.
• The DNA is mixed with the cell which are present in culture and then a
fine focus of laser beam is passed on the cell surface .
• Laser beam forms a small pore which is sufficient for uptaking of DNA
into the cell .The pore form transitory and soon repaired.

18. Nucleofection
• Nucleofection is an electroporation-based method that uses a combination
of specialized solutions and specific electric parameters to achieve direct
delivery of plasmid DNA into the cell nucleus, resulting in enhanced gene
expression.
• A new, electroporation-based transfection method that enables the DNA to
enter directly the nucleus, for the transfection of keratinocytes.

19. References
• Damjanov ,1990 Teratocarcinoma stem cells. Cancer Surv. 1990;9(2):303-
19.
• Distler,J., Jungel,A., Kurowska-Stolarska,M., Michel,B., Gay,R., Gay
S.,and Distler,O.,2005. Nucleofection: a new, highly efficient transfection
method for primary human keratinocytes Experimental Dermatology 14:
315–320.
• K.H. Khan, 2009 Gene transfer technologies leading to transgenic animals
Journal of Ecobiotechnology 1/1: 032-040.
• Martin, G. R. and Evans M.,1975. Differentiation of Clonal Lines of
Teratocarcinoma Cells: Formation of Embryoid Bodies In Vitro Proc. Nat.
Acad. Sci. USA. 1441-1445.
• Maksimenko, O. G., Deykin, A.V. Khodarovich, Yu. M. And Georgiev, P.
G. 2013 Use of Transgenic Animals in Biotechnology: Prospects and
Problems. Acta naturae 5(1):33.
• Pedro Esponda 2005. Transfection of Gametes: A Method to Generate
Transgenic Animals Int. J. Morphol.,23(3):281-284.