Specialized Chromosomes and Their Uses in Caenorhabditis elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 54

be detrimental for experiments where native or low-level expression of the transgene
is critical. RNAi-like effects such as cosuppression can also result in the suppression
of endogenous gene function, complicating analyses ( Dernburg et al., 2000 ).
3. Complex Arrays
It is the repetitive nature of transgenic arrays that is the most likely cause of their
preferential silencing in the germ line ( Kelly et al., 1997 ). To circumvent this, arrays
can be constructed in such a manner as to limit the number of reparative elements.
One way this can be done is with the addition of fragmented genomic DNA into
injection mixes ( Kelly et al., 1997 ). The incorporation of the genomic fragments acts
as a buffer to limit repeat sequence formation. These ''complex'' arrays transmit as
heritable extrachromosomal complexes in the same manner as standard arrays.
4. Advantages and Disadvantages
The major advantage of complex arrays is for analysis of germ line-expressed
genes. For unknown reasons however, germ line expression from complex arrays is
not as stable as standard arrays and expression can disappear after the first few
generations even in strains that retain the array. Maintaining animals at 25 C sup-
presses transgene inactivation ( Reese et al., 2000; Strome et al., 2001 ).
5. Biolistic Transformation
Microparticle bombardment is another method commonly used for transgenic array
transformation ( Jackstadt et al., 1999; Praitis et al., 2001; Wilm et al., 1999; Zhao
et al.,2009 ). In this approach, DNA is bound onto gold particles that are then ''shot''
into worms using a ballistic bombardment instrument or ''gene gun'' ( Wilm et al.,
1999 ). Particle bombardment generates extrachromosomal arrays with a significant
number of transformants having only a few copies of the transgene integrated at various
nonhomologous sites in the genome ( Jackstadt et al., 1999; Zhao et al., 2009 ). The
mechanism of integration is unclear ( Fire, 1986; Girard et al., 2007; Praitis et al.,
2001 ). For this technique, the transgenes must be subcloned into the same plasmid
containing the transformation marker. This is to ensure that all required transformation
and reporter components are delivered and integrated together ( Jackstadt et al., 1999 ).
6. Advantages and Disadvantages
This method while technically more difficult has several advantages over standard
injection methods. An important advantage of this approach is that stable integrated
transgenic strains can be isolated directly ( Praitis et al., 2001 ). Moreover, many
integrated transgenes do not undergo germ line silencing ( Praitis et al., 2001 ).
Finally, a scaled-up bombardment protocol has been developed to allow consistent
isolation of strains with homologous gene replacements ( Berezikov et al., 2004 ).