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

C2C12: mousemyoblast cell line

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

Lower Eukaryotes (Yeast):Origin and partitioning sequences required

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

MyoD expression correlates with potential target gene expression in transfected cells

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

MyoD is expressed only in muscle in mouse

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.