1. 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.

2. Cloning Genes Differentially Expressed in Two Cell Populations Davis, 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

4. C2C12: mouse myoblast cell line

8. 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 APPROPRIATE TO 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 INJECTED DNA).

9. Methods of transfection for Eukaryotic Cells: calcium phosphate treatment, electroporation, viruses, ballistic method, lipofection, microinjection

10. Lower Eukaryotes (Yeast): Origin and partitioning sequences required

11. 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.

14. MyoD expression correlates with potential target gene expression in transfected cells

15. Expression of MyoD in other cell types induces expression of muscle specific genes and aspects of muscle differentiation.

16. MyoD is expressed only in muscle in mouse

17. 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.