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methods

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, synthesize proteins, genomic DNA is transcribed into mRNA, which is then translated into a protein. In order to decrease or eliminate protein production , the genomic DNA coding for the mRNA can be removed, or transcription or translation can be stopped.

Removing regions of the genome that code for particular proteins is difficult. Not only are genomes difficult to alter without inadvertently damaging the organism, but genomic alterations have many limitations. For instance, the organism must have relatively few cells to effectively weed out unwanted DNA.

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

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 isoforms(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

Artificial microRNA activates a similar RNA interference mechanism but requires a much shorter input, only 21 base pairs. We used the protocol outlined [reference, link to the help page where we got the info for designing the primers for the amiRNA construc] to create a constitutively expressed amiRNA construct that is processed by the plant cell to create a short hairpin RNA (shRNA) of 21 base pairs complementary to the target gene.

Broad Overview				Detailed Descreption
AmiRNA creation broad overview   click to enlarge				AmiRNA creation detailed process   click to enlarge

In order to create our amiRNA hairpin constructs we used the plasmid RS300. RS300 contains sequences that would come together to form a hairpin and target a particular sequence in plants. Since we wanted to target our sequences instead, we used a multi-step pcr process to replace the plant's endogenous miRNA with our miRNA sequences. We created four primers, two of which contained the sequences we wanted to insert. These two primers would be used to amplify the region in between the plant's own miRNA sequence and add on "our" miRNA sequences to the ends of this region. The other two primers were used to amplify the regions before and after the plant's endogenous miRNA sequences. These three pieces were then assembled together through pcr, such that our final construct would contain a biobrick end followed by a stretch of the original RS300, followed by a stretch of ~20 base-pairs that were unique to the allergen being targeted, followed by a stretch of RS300, followed by another stretch of ~20 base pairs that were unique to the allergen being targeted, finally ending with a stretch of RS300 and a biobrick end. See images above for clarification.