Miniproject
Reverse Genetics- Gene Knockouts & Overexpression
Reverse genetics is where one has already cloned or determined the sequence of a gene and wishes to determine the function of that gene. It is a common approach with the availability of genome sequences. Although we have the genomic sequence and can make a good approximation of what proteins are encoded by the genes in the genome and their biochemical function, their function in the organism can remain largely unknown unless they are common housekeeping genes.
One powerful tool in reverse genetics is the creation of a null mutation in a gene of interest and determining the resulting change in phenotype or function of the organism. This technique uses transformation of a DNA construct to cause a deletion of or large insertion within the gene of interest that will eliminate the gene’s function. This is desirable over single base mutation since small changes may only subtly alter function or add a new different function thus masking the true function of the gene. Gene deletions or large insertions is referred to as a knock-out of the gene or gene targeting. Although this may be done on a gene-by-gene basis, the greatest resource in model genetic systems is the genome-wide knockout projects where every gene is systematically knocked-out and then made available to the research community. Obvious phenotypes might be noted, but it is left to individual researchers to use the knockouts to associate a function with the gene. Examples of large-scale gene knock-outs in model systems are:
Yeast: http://www-sequence.stanford.edu/group/yeast_deletion_project/deletions3.html
Homologuous recombination allows deletion of entire genes based upon introduction of a deletion construct. A consortium has created a knockout of the 6,000 genes in yeast have been knocked-out. Of these, 18.2% are essential for growth on rich glucose medium.
Drosophila: http://www.fruitfly.org/p_disrupt/index.html
The manipulation of specific genes and cells is more difficult for drosophila to do gene targeted recombination. Instead, a library of transposon (P-element) drosophila mutants are made. A consortium is screening such library to identify where the transposon has inserted in each line. A researchers interested in a specific gene can then identify available strains with a P-element insertion in or near their gene of interest.
Arabidopsis: http://signal.salk.edu/tabout.html
Homologous recombination is very low so insertional mutagenesis is used. Although transposons have been used, they can be unstable. Instead, transformation with a plasmid has been used for insertional mutagenesis since the 6kB plasmid is large enough to disrupt most genes and it integrates into a random location. The Salk Institute in LaJolla CA has transformed a DNA vector (T-DNA carrying a selectable marker of kanamycin resistance) randomly into Arabidopsis. Collecting different kanaymin-resistant transformants, they characterized about 18,000 independent insertions into different genes mapped by sequencing the DNA flanking the insertion in that plant. Some of these lines survive only as heterozygotes, indicating that the gene may be essential for survival.. Researchers can look up genes and find Arabidopsis lines that have an insert in or near their gene of interest.
C. elegans: http://www.geneservice.co.uk/products/rnai/Celegans.jsp
Integration of DNA is low and most DNA stays as a large extrachromosomal tangle. Thus the gene disruption method of choice is to use RNA interference. C. elegans genes has been expressed as double-stranded RNA in a separate bacterial clones. These are fed to C. elegans and the double stranded RNA turns off the respective gene in C. elegans. 16,757 genes have been expressed, representing 87% of the genome. Since RNAi reduces but does not eliminate expression of a gene, consortiums are currently creating small deletions using treatment of worms with 4,5',8-trimethylpsoralen and UV light.
Mouse: http://www.nih.gov/science/models/mouse/knockout/
Mouse genetics uses homologous recombination to specifically delete each gene. Each is expensive since the genomic manipulations are performed in embryonic stem cell cultures and these need to be reinserted into mice.
Over expression. Another way to tweak the gene so that it will alter the phenotypes it is involved in is to cause the organism to specifically overexpress that gene. This should cause a change in the phenotypes related to the cellular function where this gene is involved. It overcomes the problem of gene redundancy where a gene family contains several genes that have a similar function. Knocking-out one gene can fail to cause a change in phenotype since the other genes can compensate for the lost gene. However, if one overexpresses one gene, the related genes are less likely to compensate and hide the change. One can overexpress a gene by recombinant DNA techniques where the gene of interest is fused to a strong promoter. In Arabidopsis, the Cauliflower Mosaic Virus (CaMV) 35S promoter induces transcription strongly in most tissues. The cloned coding region of the gene of interest is fused to the CaMV 35S promoter and that DNA construct is transformed into Arabidospis. Even one copy of the overexpression construct can alter related phenotypes. An approach that performs this to random genes is called activation tagging. A T-DNA that is inserted randomly in the geneome is engineered to carry the strong enhancer elements of the CaMV 35S promoter. This is expected to cause the overexpression of genes surrounding the site of insertion. The location of the T-DNA insertion is mapped and researchers can search for lines that represent such insertion near their gene of interest.
What we will do:
We will use reverse genetics to look at several predicted genes among the approximately 26,000 genes in Arabidopsis. We will use seed supplied from the Arabidopsis stock center that corresponds to insertions in specific genes. We will grow these seed and look for phenotypes arising from the gene disruption. Identifying a phenotype will allow us to link the gene with a function.
Some Arabidopsis are carrying Knock-outs of a specific gene. Since some plants may be heterozygous for the Knock-out, not all plants will necessarily display an altered phenotype. Other lines are carrying constructs that specifically overexpress that gene because it was fused to the CaMV 35S promoter. Since this is dominant, a phenotype might be altered in all plants. However, homoxzygous plants would be expected to display a bigger change since it is overexpressing more protein with two copies of the gene.
The reference for the creation of the collection of Arabidopsis gene knock-outs is a 2003 Science article:
http://www.sciencemag.org/cgi/content/full/301/5633/653?ijkey=dtG8cC9x1Qe96&keytype=ref&siteid=sci
Procedure:
You and your partner will be given a tube of seed supplied by the Arabidopsis stock center. It will contain about 10 seeds. Write down the number from the tube. Look at the following list to find the biochemical function of your gene knock-out and how the gene was modified.
A. short-chain dehydrogenase/reductase protein - T DNA knock out
B. Encodes a microRNA (miRNA) that encodes no protein but would be expected to suppress another gene’s expression - T DNA knock out
C. zinc finger transcription factor - activation tag
D. Short 51 aa peptide- small for a protein. “DEVIL genes” with no motifs- activation tag
E. calcium-binding protein annexin - activation tag
F. No protein motifs- activation tag
G. Myb DNA binding protein - gene fused to CaMV 35S promote
H. Beta alpha alpha transcription factor- gene fused to CaMV 35S promote
I . Beta alpha alpha transcription factor as above but this construct carries an added VP16 that assures it will activate target genes - gene fused to CaMV 35S promote
J. B3 Transcription factor- gene fused to CaMV 35S promoter
K. Myb transcription factor- activation tag
Plant the seeds and observe the plants while they grow and compare them to your Arabidopsis population growing in the forward genetics project. We know the gene in which these plants are mutated and the above predicted molecular function of the gene, but we wish to find a change in the phenotype to indicate the broader function of the gene. Look for developmental differences. However, many phenotypes are conditional- they only appear under specific conditions. Look for differences in timing of the different stages of development. Look for sensitivity to stresses- turning purple with high light stress, wilting upon water stress, sensitivity to diseases.
-How many genes in Arabidopsis are in the gene family?
Find gene families in Arabidopsis at: http://www.arabidopsis.org/browse/genefamily/index.jsp.
If the gene family is not annotated in this list, go to the home page of the Arabidopsis database http://www.arabidopsis.org/index.jsp and type the domain in the search box in the upper right-hand corner. Select “protein” under the drop-down box to the right. Collect genes that have that domain in the list that you find.
-How is this protein involved in Arabidopsis function? Is there any process in plants that this function may suggest? Find the function on the web- do not limit your self to plant proteins.
As the semester progresses, we will bring in other details known about these genes, such as which tissues they are expressed in, to help focus a search for a phenotype.