**1.3 Advantages and disadvantages of BACs**

Genes expressed from BACs mirror endogenous gene expression far more accurately than other cloning systems. The large size of BACs help to minimize site of integration effects, a phenomenon which has been defined as endogenous sequences (such as gene coding regions and distal regulatory elements) to be disrupted, and to produce potentially undesirable phenotypes (Adamson, Jackson et al. 2011) in gene cloning technology. The larger sized BAC constructs contain enhancers and locus control regions, which leads to more accurate gene expression *in vivo* (Townes, Lingrel et al. 1985; Jones, Monks et al. 1995). 1995). The human genome BACs consist of the full gene structure, including untranslated regions, exons and introns, alternative promoters and splice sites and microRNA coding sequences. RNAs such as RNA splicing or microRNAs play very important role in gene regulation (Jackson and Standard 2007). Therefore the human genome BACs will ensure full mRNA processing and splicing when genes are transcribed, and produce the full complement of protein isoforms once mRNAs are translated. BACs can be transfected and expressed in mammalian cell lines although transfection efficiency and copy numbers are low (Magin-Lachmann, Kotzamanis et al. 2004; Sparwasser and Eberl 2007).

BACs also have a number of disadvantages. A construct containing a large genomic fragment is likely to contain non-related genes that may lead to indirect, non-specific gene expression and unanticipated changes in the cell phenotype; Secondly, compared to plasmids or other gene expression vectors, the generation and screening of recombinant BAC constructs can be time-consuming and labor-intensive. Also, the oversized BAC DNA constructs are more easily sheared and degraded during manipulation before transfection; and some random recombination events may occur, for example, LoxP sites may lead to random Cre-mediated recombination (Semprini, Troup et al. 2007). Finally, repeating homologous sequences in some BACs constructs may undergo intramolecular rearrangements, which reduce the recombination efficiency and increase the rate of falsepositive clones in some selection/counter-selection approaches (Narayanan 2008).

Overall, BACs have numerous advantages when compared to conventional plasmids. They protect the gene from site of integration effects and produce accurate regulation of transcription and translation. However, the large size results in technical difficulties when handling them as well as the potential non-specific gene expression. Therefore the application of BACs as a gene expression model system should be careful considered based on the pros and cons previously described.
