Transcript No Slide Title

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 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
Hypothesis: 5-Azacytidine treatment of 10T1/2 cells
activates a gene that programs cells to differentiate
muscle
Use DIFFERENTIAL (or SUBTRACTIVE) HYBRIDIZATION
isolate cDNA for this gene
5-azaC myoblasts (or C2C12)
10T1/2 cells
mRNA
mRNA
P-32 cDNA
hybridize and remove double-stranded material
C2C12 mRNA
(or aza myoblast)
SUBTRACTED P-32 cDNA
negative
(
selection
)
hybridize and select DOUBLE STRANDED material; RNaseH treatment
Subtracted P-32 cDNA
positive
(
selection
)
 cDNA library prepared from myoblasts
Use these as probes to screen
C2C12: mouse
myoblast 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 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).
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
DNA
DNA remains cicular transiently
is imported to the nucleus
input
Input DNA is converted into long tandem repeats
chromosome
DNA becomes integrated RANDOMLY into host chromosome
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.