Student Reader

Gene Control in Development: Laboratory Techniques

Visualizing Differential Gene Expression

In Situ HybridizationShows
Antibody Staining
Microarray Analysis
Direct RNA InjectionThis entails injecting RNA into embryonic cells and then observing the outcome. This technique requires large embryos, large cells and external development.

Studying Gene Function

RNA InterferenceRNA interference can phenocopy a mutation by blocking action of a specific gene.
MorpholinoMorpholinos block function (usually translation) of complementary mRNA. They are generally short, with about two dozen nucleobases linked by phosphorodiamidate linkages with a backbone of neutral morpholine rings. Their short length and ability to interfere with gene expression may seem similar to siRNA. However, a morpholino's structure is most analogous to a ssDNA oligonucleotide, except with different backbone and linkage molecules. Sometimes morpholinos are paired to partially complementary DNA so they can be transfected like normal dsDNA. This hybrid oligonucleotide is prepared at a very high morpholino:complement ratio to allow the assumption that all the DNA complement has been bound (albeit tenuously). Once transfected into a cell, the morpholino can readily disassociate and perform its function.
siRNASmall interfering RNA (siRNA) is a type of dsRNA found in the RISC complex. The RISC Complex activates RNAse to degrade mRNAs sharing the same sequence as the siRNA.
Genetic ScreenGenetic screens involve mutagenizing a male's sperm (making some m/+) and mating it with a wild-type female (+/+). Linkage analysis can pinpoint the gene carrying the mutation. The F1 progeny will rarely carry a mutation in the gene of interest. A mutant male F1 is mated with a wild-type female and ½ of the F2 progeny will carry the mutation. Mating the F2 progeny will result in ¼ of progney being homozygous for the mutation. The best model organisms for a genetic screen have many progeny, small size and external development -- Drosophila and Zebrafish are ideal.
Transgenics
  1. Fertilized Egg (2 haploid pronuclei).
  2. Microinject DNA into male pronucleus.
  3. One-cell embryo with diploid nucleus.
  4. Transfer to oviduct of recipient female.
  5. Transgenic mouse results.

You can add a DNA construct engineered with a fly eye-formation gene attached to a leg promoter. The result is that an eye forms on the fly's leg. Alternatively, you can add a DNA construct carrying a dominant-negative mutation; the mutant gene product interferes with the wild-type gene product to cause loss of function.

Specific MutationsMutations can be made in specific genes by engineering a vector to contain the gene of interest interrupted by a marker (ie, Neomycin resistance). After screening for the marker, homologous recombinants are identified by Southern Blot.

Studies

"How To" Make a Special Delivery Morpholino Oligo

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