Enhancer Analysis of the a1(II) and a2(XI) Collagen Genes in Transfected Chondrocytes and Transgenic Mice - Methods in Molecular Biology: Extracellular Matrix Protocols - page 185

expression transiently of transfected DNA constructs; the amount and purity of
DNA, the ratio of DNA to transfection reagent, the cell density at time of trans-
fection, and the transfection time may require optimization for each construct or
cell type.

5. If CAT activity is to be normalized to DNA content, rather than protein content,

centrifuge the sample prior to heat inactivating. Save the cell pellet for DNA
content analysis and use supernatant for CAT analysis.

6. Cotransfection with a control plasmid utilizing a different reporter gene is

required to determine the transfection efficiency between experimental groups.

7. It is important to perform preliminary cotransfection experiments to optimize

both the amount of vector DNA and the ratio of coreporter vectors added to the
transfection mix.

8. The pRL family of coreporter vectors for cotransfection assays are useful to opti-

mize transfection efficiency. However, promoter activity of the control coreporter
gene may affect activity of the promoter and enhancer of the test gene.

9. Background staining of the endogenous -galactosidase activity can be decreased

by staining tissues at pH 7.4. Inclusion of an intron cassette at the 5' leader
sequence and poly-A signal at the 3' end of the construct often increases reporter
gene activity probably because these sequences stabilize mRNA for the transgene.

β

10. Activity of the endogenous -galactosidase gene may increase in certain tissues

(e.g., bone and kidney) in late developmental stages and cause increased
background staining. Green fluorescent protein (GFP) may also be used as a
reporter gene.

β

11. If the level of reporter gene expression is low in G0 embryos, homozygous

transgenic mice can be established and analyzed. Higher levels of expression of
the reporter gene can be obtained in these mice because of double dosages of the
transgene in the homozygous mouse.

References
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