Gene Transfer

Manita Aryal

Introduction
to transfer a gene from one DNA molecule to another DNA molecule 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

Enzymes Used in Recombinant DNA Technology

Enzyme: Function Type II restriction endonucleases : Cleave DNAs at specific base sequences DNA ligase: Joins two DNA molecules or fragments DNA polymerase I (E. coli): Fills gaps in duplexes by stepwise addition of nucleotides to 3 ends Reverse transcriptase: Makes a DNA copy of an RNA molecule

Enzymes Used in Recombinant DNA Technology

Polynucleotide kinase: Adds a phosphate to the 5-OH end of a polynucleotide to label it or permit ligation Terminal transferase: Adds homopolymer tails to the 3-OH ends of a linear duplex Exonuclease III: Removes nucleotide residues from the 3 ends of a DNA strand Bacteriophage exonuclease: Removes nucleotides from the 5 ends of a duplex to expose single-stranded 3 ends Alkaline phosphatase: Removes terminal phosphates from either the 5 or 3 end (or both)

Gene transfer methods

Normally include three categories: 1. transfection by biochemical methods 2. transfection by physical methods 3. virus-mediate transduction

Techniques for the gene transfection

(1) Lipid-mediated method (2) Calcium-phosphate mediated (3) DEAE-dextran-mediated (4) Electroporation (5) Biolistics (gene gun) (6) Viral vectors (7) Polybrene (8) Laser transfection (9) Gene transfection enhanced by elevated temperature

Plasmid

Ti Plasmid
T-DNA region
DNA between L and R borders is transferred to plant as ssDNA;
Tumorproducing genes

Opine catabolism Virulence region ORI

T-DNA encoded genes can be substituted by target genes

Ri Plasmid

Ti-plasmid based vectors

Binary systems
Needs 2 vectors: Disarmed Ti plasmid with gene of interest (no vir genes) Helper vector for infection (with vir genes)

Co-integrated vectors
Needs 3 vectors
Disarmed Ti plasmid capable for infection Intermediate vector with T-region and gene of interest (transferred by conjugation) Helper vector for transfer of intermediate plasmid into A.tum

Form co-integrated plasmid after homologous recombination on T-DNA

Hybrid Ti Plasmid

Gene Transfer using Agrobacterium

Gene Transfer using Agrobacterium

Gene Transfer using Agrobacterium

Virus as vector

Virus as vector

Thank you