REVERSE GENETICS

Reverse Genetics

Gene silencing and homologous recombination are two commonly used approaches used for targeted gene mutation, in contrast to non-targeted disruptions of genes achieved by transposon mediated and chemical mutagenesis. For such a model plant as Arabidopsis, T-DNA insertion mutants have been produced and are available for researchers(Krysan et al., 1999). It should be noted that transpositions are not completely random(Krysan et al., 2002) and thus mutation of all genes is not guaranteed. However the mutants are a valuable research tools for understanding the function of the gene. The required T-DNA insertion mutant can usually be ordered and detailed phenotypic analysis performed.

Two novel approaches of reverse genetics:

 1) targeted gene silencing by RNA interference and

2) TILLING (Targeting Induced Local Lesions in Genomes) – a recently developed reverse genetics technique.

Targeted gene silencing

Gene silencing by RNA interference (RNAi) is one of the most exciting breakthrough of the past decade in functional genomics and promises to be a very useful instrument for therapeutic gene silencing. The phenomenon of RNAi was first discovered during experiments associated with changes in pigmentation in the petunia plants. Introducing extra copies of a pigment biosynthesis gene did not increase the color intensity in the flower as was expected, but the flowers became less colorful than the wild flowers (de Lange et al., 1995; Hannon, 2002; Bushman, 2003; Foubister, 2003). The term RNAi was first introduced after Andrew Fire and Craig C. Mello discovered that injection of dsRNA into the nematode worm Caenorhabditis eleganscause the specific silencing of genes highly homologous to the supplied sequence (Fire et al., 1998; Elbashir et al., 2001). Double-stranded RNA (dsRNA) triggers the RNAi process and can be endogenous or exogenously introduced into the cells(Shuey et al., 2002).The basis of the RNAi process, production of the functionally similar endogenously produced siRNAs, is quite similar in many organisms and the enzymes required for this process show high inter species homology (Hamilton and Baulcombe, 1999; Paddison et al., 2002). Processing of dsRNA precursors into small interfering RNAs (siRNAs) is mediated by special dsRNA-specific RNase-IIItype endonucleases, known as Dicer. This results in formation of 21-25 nucleotide double stranded RNA duplexes with symmetric 2-3 nucleotide 3’ overhangs, which are called small interfering RNsiRNA. The siRNAs are afterwards incorporated into the RNA-induced silencing complex (RISC), where an RNA helicase unwinds the inactive double-stranded siRNA, converting it to an active single-stranded form (Nykanen et al., 2001; Hannon, 2002; Plasterk, 2002). Nevertheless only one strand, known as the guide strand is stabilized in RISC complex, while the passenger siRNA strand is degraded. (Gregory RI., et al., 2005). An active RISC complex uses the guide siRNA to find and destroy the complementary sequence of mRNA, causing in turn gene silencing (Bushman F, 2003: 49, Nykänen A., et al., 2001). In plants in contrast to other organisms miRNA have perfect or near perfect complementarity to their targets(Axtell et al., 2011). Thus plant siRNAs are easily designed.

RNA interference (RNAi)

TILLING (Targeting Induced Local Lesions in Genomes)

Generation of mutated lines by transposons, TDNA or RNA interference is technically difficult in some organisms.The difficulty comes due to the lack of an efficient transformation system and due to the large genome for some organisms like barley (Bennett and Smith, 1976; Ahringer, 2006; Chawade et al., 2010). One way to increase variation in the breeding process would be to use radiation or chemical mutagens such as EMS (ethyl methanesulfonate). The mutagenic substance EMS preferentially alkylates guanine bases. The resulting O-6-ethyl guanine paired with cytosine is misread by the DNA-replicating polymerase which insertsa thymine residue instead of a cytosine residue. This results in G-C base-pairs (bps) being mutated to AT (Hoffmann, 1980). Mutations in coding regions can be silent, missense or nonsense and mutations in non-coding promoter or intron regions can result in up- or down-regulation of transcription (Rose and Beliakoff, 2000). TILLING (Targeting Induced Local Lesions in Genomes) is a recently developed reverse genetics technique, based on the use of a mismatch-specific endonuclease (CelI), which finds mutations in a target gene containing aheteroduplex formation (Henikoff et al., 2004; Gilchrist et al., 2006; Chawade et al., 2010). If the mutation frequency is high and the population size large enough, mutated alleles of most, if not all, genes will be present in the population. The technique involves PCR amplification of the target gene using fluorescently labeled primers, formation of DNA heteroduplex between wild type and mutant alleles (PCR products, corresponding to the mutant and wild type alleles are heated and then slowly cooled), followed by endonuclease digestion specifically cleaving at the site of an EMS induced mismatch. The sizes of the amplicon cleavage fragments are often analyzed by a Li-COR (McCallum CM, 2000 ) or MALDI-TOF (Matrixassisted laser desorption/ionization-time-of-flight mass spectrometer) (Chawade et al., 2010) system. It is possible to apply TILLING to genetically complicated crops, such as wheat for example (Slade et al., 2005).

One of the greatest benefits of the TILLING approach is that it does not involve genetic manipulations, that results in Genetically Modified Organisms (GMO), which are not legal for agricultural applications in many countries.

Combining the Power of Forward and Reverse Genetics

Both forward and reverse genetics have their relative strengths and weaknesses. A powerful tool therefore is to consider doing both simultaneously. In TILLING projects, the first example of this comes from Lotus japonicus where Perry and colleagues at the John Innes Centre created a phenotype indexed population for reverse genetics (Perry et al. 2003 ). When performing reverse genetic screens in genes hypothesized to be involved in root symbiosis, they were able to recover useful genic lesions from a sub-population expressing root phenotypes. Typically one would screen the entire mutant collection to recover such alleles. In this example, however, the alleles were recovered in a set of 275 plants rather than the entire population of 3697, providing over a 10 fold reduction in the effort of mutation discovery. Other examples can be found for barley , tomato , pea, brachypodium and flax

(Dalmais et al. 2008 ; Menda et al. 2004 ; Talame et al. 2008 ). Similar resources exist for other reverse genetic projects such as the soybean fast neutron work (Bolon et al. 2011 ). The availability of fully genotype and phenotype indexed mutant databases provides an excellent tool for rapid gene function studies and thereby in developing effi cient strategies for the introgression of useful alleles in breeding.