Analysis of Regulatory Signals by Gene Transfer

Introduction

Necessary components of a segment of DNA to be transferred into cells: need the gene itself; 5' flank / exons / introns / 3' flank; or cDNA = copy of the mRNA (no introns) has 5' and 3' flank

Essential Elements of DNA mediated Gene Transfer

Cloned segment of DNA (including 5' and 3' flanks)

A cell line – eukaryotic cells able to propogate in tissue culture Þ defined medium for growth; 1 – 2 antibiotics

Method of introducing the cloned DNA into intact cells

Transfection Þ direct injection; viral vectors; Calcium Phosphate Precipitated form (Fig 1)

Method of Transfection

Calcium Phosphate Precipitation

Calcium Phosphate precipitates the DNA onto the cells and some of it will be taken in by the cells (low %)

Liposome Mediated Transfer (Fig 3)

Lipids surround the DNA and the liposomes will fuse with the cell memb and deliver the DNA into cells

Injection

DNA is microinjected into the nucleus Þ direct delivery Þ need big cells

Viral vectors

Use the virus to transport the DNA into the cell

Ex: retroviral vector Þ the DNA becomes incorporated into the host; 2 fates Þ stable transformant (DNA integrated) or transient transfection (DNA not integrated)

Fate of the Foreign DNA in Transfected Cells

Efficiency of transfection is low thus need a selectable marker to detect transfection occurrence

Antibiotic resistance

neomycin Þ inhibits euk cell growth Þ death

neomycin resistant gene Þ if uptaken, will grow in presence of the antibiotic

Detection of Hormone Responsiveness Elements in Cloned Genes (Fig 5)

Use of reporter genes Þ readily assayable gene

Reverse Genetics and Site Directed Mutation

creates mutations within a sequence of interest and tests the effects of that mutation in transfected cells

make mutation Þ observe phenotype

Growth hormone gene

reporter genes: CAT, Chloramphenical acetyl transferase; luciferase (glowing protein)

Transfer of Foreign Genes into Mice (transgenic mice)

Generate mice with specific genes stabley integrated into their germline DNA Þ able to pass on to future generations

Isolate fertilized egg Þ inject DNA into male pronucleus Þ put fertilized egg back into mouse Þ pups (baby mice)

Ex: rat growth hormone gene

Producing transgenic mice permanently changes the germline

Gene "knock outs" in mice

performed in embryonic stem cells which can be injected into early embryo and implanted into a mouse