biotechnology smt.g.indravathi msc,m
TRANSCRIPT
Smt.G.Indravathi MSc,M.Phil
KVR Govt. College for Women (A), Kurnool
Email. Id : [email protected]
Biotechnology
Learning Objectives
Biological Methods of Gene Transfer
In Plants- Agrobacterium mediated gene transfer
In Animals- Embryonic stem cell Method
In Animals- Retroviral method
Advantages & Limitations
Gene Transfer
Direct Gene Transfer - Physical & Chemical Methods
By using instrumentation and chemicals desired gene is transferred
directly in to the target cell.
Indirect Gene Transfer – Biological Methods
The desired gene is transferred in to the target cell by the support
of living organisms like bacteria and virus
Biological Method of Gene Transfer
Plants:
Agrobacterium mediated Gene transfer
Animals :
Retroviral mediated gene transfer
Embryonic Stem Cell method
Agrobacterium as Natural Genetic Engineer
Agrobacterium tumefaciens is a rod shaped, gram negative bacteria naturally
found in the soil that causes crown gall disease in dicot plants .
During the infection process, the Transfer DNA (T-DNA) located in Tumor-
inducing (Ti) plasmid of Agrobacterium is transferred into the nucleus of the plant
cell causing growth of tumors in the basal part of the stem (Crown region).
Thus the study of crown gall disease paved the way for using Agrobacterium in
the transfer of foreign genes by genetic manipulation.
Agrobacterium as a broad host range capacity and used for transformation in
organisms like dicots, monocots, gymnosperms and even fungi.
Bioinspiration
Crown Gall Disease Natural Ti Plasmid
Source-https://www.ag.ndsu.edu/drappletree/crown-gall-CC BY-SA-NC Source: http://en.wikipedia.org/wiki/Transfer_DNA (CC)
Modified Ti - plasmid
Wild type Ti plasmid from Agrobacterium cannot be directly used as vector
system due to their large size, presence of tumor causing genes and absence of
marker genes and unique restriction sites within T-DNA region.
Most of the T-DNA region of bacterial plasmid is replaced with the gene of
interest while leaving the left and right border sequences.
The T-DNA region is defined not by its sequence but by its borders which
enables its insertion into host plant genome.
Therefore, Ti plasmids are genetically engineered to incorporate the DNA
sequences necessary for effective transformation of plant cells.
Modified Ti plasmid
Source- http://www.patentlens.net/daisy/AgroTran/g1/850.html (CC)
Source: http://illl.du.ac.in/- Namrata Dhaka, Department of Genetics, University of Delhi, South Campus.
EXPRESSION CASSETTE
Steps involved in Agrobacterium mediated Gene transfer
Identification and Isolation of Cry Gene – Bacillus thuringenesis
Insertion of Cry Gene in a Ti plasmid Vector
Introduction of the recombinant vector in an Agrobacterium host
Screening of transformed Agrobacterium cells
Co- cultivation of host cells with cotton plant material(callus).
Production of a transgenic Cotton Plant called Bt Cotton.
Agrobacterium-mediated transformation
http://omicsgroup.org/journals/current-status-of-sugarcane-transgenic-an-overview-2169- 0111.1000112.php?aid=18824 (CC)
Agrobacterium mediated Gene transfer for BtCotton
Source- https://ipkitten.blogspot.com/2016/03/a-monsanto-case-that-could-alter.html- CC BY
Insecticide Resistant Plants
Steps involved in ESC mediated Gene transfer
Embryonic stem cells (ESC) are cell line isolated directly from inner cell mass
(ICM) of early mouse embryos of blastula stage.
The efficiency of genetically manipulated ESC is maintained even after
differentiation at tissue and organ level. ESC mediated gene transfer is most
common in mice.
ES cells are transformed by exposing cultured cells to the foreign DNA by
Microinjection which integrates into the mouse genome, by means of
"homologous recombination”
: Embryonic stem cell mediated transfer
Source-molecular biotechnology: principles and applications of recombinant DNA by Glick et al., 2010
Steps involved in ESC mediated gene transfer
Successfully transformed ES cells are then selected and injected
into cavity of host blastocysts of different mouse strain and re-
implanted into uterus of foster mother (pseudo-pregnant mother).
Expression of transgene is tested by analyzing the tissue of animals
produced in F1 generation and it acts as the transgenic founder.
Since these ES cells colonizes host embryo , they contributes to the
germ line.
Retroviral vectors❑ The viral genes, gag, pol and env are responsible for producing specific proteins that
directly relate to viral structure and replication. These genes are replaced with the
transgene of interest .
❑ At each end of the retroviral DNA are long terminal repeats (LTRs) which enable the
virion to integrate itself in to the host genome.
❑ The LTR sequences contain promoter and enhancer sequences that can be modified
to promote expression of the transgene.
Retrovirus mediated Gene transfer
Retroviral Mediated Gene Transfer
This gene transfer technique is mediated by means of a carrier such as
retrovirus.
Foreign genes are delivered to host cell by transfection method. In this
method, pathogenic viral genes are replaced by transgene.
The method allows insertion of small size DNA inserts upto 8 kb
The main advantage of this method is that it does not lead to abortion
and the probability of expression is much better.
Steps involved in Retrovirus mediated Gene transfer
4 to 8 cell stage early embryos are taken for retroviral infection.
These embryos are infected with Recombinant retrovirus containing
transgene
Retroviruses, carrying RNA as its genetic material is “reverse transcribed”
to produce DNA, which integrates itself into host cell and result in chimera.
Transmission of virus gene is only possible if retrovirus had integrated
transgene into the germ cells .
Expression of transgene is tested by analyzing the animals produced in F1
generation and it acts as the transgenic founder.
Retroviral Mediated Gene Transfer
Source-molecular biotechnology: principles and applications of recombinant DNA by Glick et al., 2010
Conclusion
Biological gene transfer – The target cells are transformed with the support
of biological agents in the form of vectors/host cells
In Plants- Agrobacterium as a broad host range capacity and used for
effective transformation in dicots, monocots, gymnosperms and even fungi.
In Animals – ES cells and retrovirus integrates transgene into the germ
cells , they contributes to the germ line.
Expression of transgene is tested by analyzing the tissue of organisms
produced in F1 generation and it acts as the transgenic founder.
Smt.G.Indravathi MSc,M.Phil
KVR Govt. College for Women (A), Kurnool
Email. Id : [email protected]
Thank You