Gene Control in Development: Laboratory Techniques
By Levi Clancy for Student Reader on
updated
- Genetic techniques
- 5'-Deletion Mutants
- Ames Test
- Cloning Vectors
- Conjugation
- DNA Fingerprinting
- DNA Miniprep
- Gel Shift Assay
- Gene Control in Development: Laboratory Techniques
- Gene Targeting
- Genetic Engineering
- Genetic screen
- In Vitro Nuclear Run-on Experiment
- Interrupted Mating Experiment
- Knockout mutation
- Linkage analysis
- Polymerase Chain Reaction
- Promoter (Transcriptional) (RNA) Fusion
- Reporter Gene
- Restriction Enzymes (Endonucleases )
- Sequence Alignment
- Shotgun sequencing
- Temperature Sensitive Mutant Experiment
- Transformation
- Transgenes
- Translational (Protein) Fusion
- Transposon Tagging
- cDNA Microarray
Visualizing Differential Gene Expression
| In Situ Hybridization | Shows |
|---|---|
| Antibody Staining | |
| Microarray Analysis | |
| Direct RNA Injection | This 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 Interference | RNA interference can phenocopy a mutation by blocking action of a specific gene. |
|---|---|
| Morpholino | Morpholinos 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. |
| siRNA | Small 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 Screen | Genetic 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 |
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 Mutations | Mutations 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. |