• home
• project
  • vectors
  • allergy
  • flavor
  • color
  • genetic fence
  • human practices
  • safety
  • parts
• results
• people
  • students
  • teaching fellows
  • advisors
  • acknowledgements
  • gallery
• online
  • blog
  • twitter
  • openwetware
  • facebook
  • contact us

allergy knockdown allergen targets lab notebook methods parts and primers references

methods

As proteins provoke the majority of allergic reactions, we hope to diminish the allergenic properties of our plants by suppressing the expression of proteins that aren't necessary for the plant's survival but have been found to cause allergic responses in humans. This problem is complicated by the fact that proteins with allergenic properties may have several slightly different (but similar enough to remain allergens) homologues spread throughout the plant's genome. Subject to the constraint of the plant's survival, we would like to reduce or eliminate the expression of these homologues as well.

New version:

Creating hypoallergenic plants is a complicated process. Many proteins that provoke allergic reactions are essential for the plant's survival, and plants frequently produce more than one version of the protein. Our ability to reduce and eliminate allergy-inducing proteins from a plant is constrained by what proteins the plants need for survival and our success in eliminating homologous versions of the offending protein.

When plants, or any organism, synthesizes proteins, genomic DNA is transcribed into mRNA, which is translated into a protein. In order to reduce or eliminate production of proteins in any organisms, either the genomic DNA coding for the mRNA is removed, or transcription or translation has to be stopped.

Removing the region of the genome coding for the protein is difficult for many reasons, one of which is that genomes are difficult to alter without inadvertently damaging the organism. Another difficulty is that genomic alterations have many limitations. For instance, the organism must have relatively few cells to effectively weed out unwanted DNA.

The preferred method of stopping protein production in plants is through a process called RNAi, short for RNA interference. The general concept behind RNAi is using special types of RNA to stop the translation process.

RNAi

RNAi (RNA interference) is a process used to control expression of genes in living cells. It down-regulates gene expression by preventing the translation of specific proteins. In this process, the cell's machinery recognizes double stranded RNA sequences present in the cell. These sequences are then cut up into shorter fragments. mRNA transcripts that are complementary to these shorter sequences are then cleaved, thereby preveting trasncription of the proteins that would have come from these sequences. By introducing synthetic double stranded RNA sequences complementary to the sequences of the various allergens that we would like to target, we hope to knockdown the expression of these allergens and their homologues (versions of these allergens with a similar sequence that would still produce an allergic response).

hpRNA

With RNAi, the problem of creating a hypoallergenic plant reduces to the problem of integrating short RNA strands into the RISC, each with complementarity to RNA which ribosomes would otherwise transcribe into allergenic proteins. One mechanism of flagging RNA for RISC-incorporation is to place the RISC-targeting sequence (~300bp), an intron-specific sequence (200-, and the reverse complement of the RISC-targeting sequence (~300bp) under a promoter (in nature these constructs could also be found in an intron). Upon transcription, this construct will form a hairpin: the targeting sequence and its reverse complement will anneal to each other while the intron-specific sequence will form the hairpin's loop. This structure is called a hpRNA, short for "hairpin RNA." The RISC will then process and incorporate part of one of the legs of the hairpin (targeting sequences) with which it will search for and destroy complementary RNA sequences.

amiRNA

The process of hairpin RNA (hpRNA) incorporation involves customizing a small (~21bp) portion of one of the hairpin legs which will actually be used for RISC specificity. Since the usual processing of hpRNA isn't entirely deterministic we can profitably bypass part of the processing, effectively jumping in to the hpRNA pipeline at a later stage that gives us more control over the sequence which the RISC will use for specificity.