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TRANSFECTION

By : Mr. Sumit Sharma

Department of Biotechnology

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CONTENT

Introduction To Transfection
Transfection Terminology
Transfection Workflow
Factors influencing transfection efficiency
Calcium phosphate precipitation
DEAE –Dextran- mediated transfection
Lipofection
Electroporation
Retroviral infection
Microinjection
Promoters
Expression vector

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INTRODUCTION TO TRANSFECTION

Transfection is the process of artificially introducing nucleic acids (DNA or RNA ) into cells, utilizing means of other than viral infection.

Introduction of foreign DNA into cells either by physical (electroporation) or chemical (cationic lipid or calcium phosphate reagents) methods.

In transfection, the introduced nucleic acid may exist in the cells transiently, such that it only expressed for a limited period of time and does not replicate, or it may be stable and integrated into the genome of the recipient, replicating when the host genome replicates.

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TRANSFECTION TERMINOLOGY

The terminology used for various gene delivery systems has evolved to keep pace with technology advances in the field and further refined to distinguish various methods and cell types.
Cell that have incorporated the foreign DNA are called transfectants.
Stable transfectants: Cells that have integrated foreign DNA in their genome.
Transient transfectants: Foreign DNA does not integrate in the genomebut genes are expressed for limited time (24-96 hours).

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TRANSFECTION WORKFLOW

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FACTORS INFLENCING TRANSFECTION EFFICIENCY

Cell type
Cell health
Confluency
Serum
Time
DNA quality & quantity

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COMMON TRANSFECTION METHODS

Calcium phosphate Precipitation
DEAE – Dextran mediated transfection
Lipofection
Electroporation
Retroviral infection
Microinjection

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CALCIUM PHOSPHATE PRECIPITATION

This method is popular transfection method since its introduction in the early 1970s by Graham and Ven der Eb, 1973.
This technique is easy and effected with many types of cultured cells
It can be used for both transient and stable transfection of variety of cultured cell types.
Principle:
DNA is mixed with calcium chloride
Addition to buffered saline/phosphate solution and incubating at room temperature
Formation of DNA- calcium phosphate coprecipitation which adhere to surface of cells
Uptake presumably by endocytosis or phagocytosis.

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METHOD USED

Steps –
Solutions used are DNA in calcium solution and 2x Hanks buffered saline solution
Add solution DNA in calcium to hanks buffered saline solution
Incubate 20-30 min . Apply the solution to a subconfluent cell culture
Incubate 2-12 hr. Replace the solution with complete growth medium.
Assay for transient gene expression or begin selection for stable transformation time.

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ADVANTAGES & DISADVANTAGES

ADVANTAGES-
Easily available
Inexpensive
Can be used for transient and stable transfection
Can be applied to wide range of cell types
Calcium phosphate appears to provide protection against intracellular and serum nuclease
Disadvantages-
Low efficiency
It is not suited for in vivo gene transfer to whole animal
Toxicity, especially to primary cells
Size and quality of precipitate are crucial to the success of transfection.

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DEAE-DEXTRAN- MEDIATED TRANSFECTION

DEAE- dextran was the first non-viral transfection method verified by Vaheri and Pagno in 1965.
DEAE –Dextran is a cationic polymer that tightly associated with negatively charges nucleic acids.
Principle –
DNA is mixed with DEAE-dextran
DNA/polymer complex comes into contact with the negatively charged membrane due to excess of positive charge contributed by polymer entry in to the cytoplasm via endocytosis or osmotic shock induced by DMSO or glycerol.
Uptake presumably by endocytosis.

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