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LECTURE 4. SCREENING cDNA LIBRARIES to ISOLATE NEW GENES:
DIFFERENTIAL HYBRIDIZATION
ORIGINAL ARTICLE:
**Davis, RL, Weintraub, H, and Lassar, A. 1987. Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell 51:987-1000.
Cloning Genes Differentially Expressed in Two Cell PopulationsDavis, RL, Weintraub, H, and Lassar, A. 1987. Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell 51:987-1000.
Muscle Differentiation 5-azacytidine(inhibitor of CpG methylation)
remove serum
Hypothesis: 5-Azacytidine treatment of 10T1/2 cells activates a gene that programs cells to differentiate into muscle
Use DIFFERENTIAL (or SUBTRACTIVE) HYBRIDIZATION to isolate cDNA for this gene
5-azaC myoblasts (or C2C12) 10T1/2 cells
mRNA mRNA
P-32 cDNA
hybridize and remove double-stranded material
SUBTRACTED P-32 cDNA (negative selection)C2C12 mRNA(or aza myoblast)
hybridize and select DOUBLE STRANDED material; RNaseH treatment
Subtracted P-32 cDNA (positive selection)
Use these as probes to screen cDNA library prepared from myoblasts
TRANSFORMATION/DNA-MEDIATED TRANSFECTION: THE ABILITY OF
NAKED DNA TO ALTER THE GENOTYPE/PHENOTYPE OF A CELL
ALL CELLS CAN BE TRANSFORMED RELATIVELY EASILY.
REGULATORY ELEMENTS APPROPRIATETO THE ORGANISM ARE REQUIRED
E.G., INJECTION OF DNA INTO THE HEART OF RATS RESULTS IN TRANSIENT TRANSFORMATION OF HEART CELLS (THAT IS THEY EXPRESS THE INJEC TED DNA).
Methods of transfection for Eukaryotic Cells:calcium phosphate treatment, electroporation, viruses,ballistic method, lipofection, microinjection
Mammalian Cells
1) Any DNA will be incorporated into the host genome: HETEROLOGOUS RECOMBINATION=NO HOMOLOGY REQUIRED. Frequency is about 0.1-1 in 1000 for most cell types. In 1 cell mouse embryos the rate is 1 in 5 when DNA delivered by microinjection.
2) Foreign DNA is incorporated in host chromosomes in a RANDOM manner. Exception: some viral vectors, if viral proteins are supplied in trans (e.g. Epstein-Barr virus vectors).
3) HOMOLOGOUS RECOMBINATION CAN OCCUR, BUT THE FREQUENCY IS MUCH LOWER (1:1-10 million) . A cell will undergo either HETEROLOGOUS OR HOMOLOGOUS RECOMBINATION, BUT NOT BOTH SIMULTANEOUSLY.
Fate of Genes Introduced into Mammalian Cells: Heterologous Recombination
Input DNA is converted into long tandem repeats
DNA is imported to the nucleus
DNA becomes integrated RANDOMLY into host chromosome
input
chromosome
DNA remains cicular transiently
Expression of MyoD in other cell types induces expression of muscle specific genes and aspects of muscle differentiation.
MyoD is related to a known family of transcription factors: the Helix-Loop-Helix (HLH) family
Subsequently, the MyoD HLH region was used to isolate cDNA clones for two closely related HLH factors, myf5 and myogenin by low stringency hybridization. All three genes are necessary for correct muscle differentiation in vivo, as demonstrated by mouse "knockout" studies.