18 cDNA Libraries
281
Principles of vector selection: expression vs. non-expression libraries
The number of vector-host systems available for cloning genes is constantly
increasing. Since this process is likely to continue, it is more appropriate for
a review such as this to discuss some of the basic principles of vector selection for cDNA libraries. It is anticipated that the readerwill then be able to
use his/her knowledge to evaluate the advantages of new systems.
Some of the factors that govern vector/host selection for cDNA library
construction are similar to those for genomic libraries. High transfection/
transformation efficiencies, stable propagation and selectable markers are
desirable. Unlike genomic libraries, the size ofthe insert is less often a problern since most fulllength cDNAs can be easily cloned into either plasmid
or lambda bacteriophage, and virtually all cDNA libraries are constructed
using one of these vectors. Historically, bacteriophage were preferred because packaging and transfection efficiencies far exceeded plasmid transformation rates and yielded a higher number of primary recombinants per
microgram of starting cDNA. Technical improvements (electroporation
and transformation of plasmids into high-efficiency competent cells)
have since made this difference negligible, and other features are now
used to select an appropriate system. Bacteriophage libraries are easier
to screen and are more likely to include Ionger gene sequences. Furthermore, one can use a modified bacteriophage which requires a minimum
insert size that is greater than the average transcript size ( eg. > Skb) to select
for Ionger cDNAs. On the other hand, plasmid libraries have some other
advantages. They are easier to amplify in a way that maintains the relative
proportion of clones within them. Plasmid DNA also is more easily prepared and transfected into eukaryotic cells for expression studies.
The most important factor that determines selection of a vector is how
the library is to be screened. As discussed previously, clones encoding genes
of interest can be identified in cDNA libraries using standard hybridization
protocols. A major advantage of cDNA libraries isthat one requires neither
the gene sequence nor a sub-fragment to isolate its fulllength clone. Because
cDNA clones are derived from mRNA, they potentially encode the protein
products of the genes from which they were derived. This feature allows an
investigator to identify genes on the basis of functional properties rather
than sequence. Antibodies, ligands, and functional attributes such as transport activity or ability to rescue function become additional tools that are
useful in identifiying clones derived from genes of interest. A yeast-based
technique, known as the two-hybrid system, has been developed to screen
for genes which encode proteins capable of interacting with a protein of
interest (bait). The assay is based on the abilitytoseparate eukaryotic tran-
281
Principles of vector selection: expression vs. non-expression libraries
The number of vector-host systems available for cloning genes is constantly
increasing. Since this process is likely to continue, it is more appropriate for
a review such as this to discuss some of the basic principles of vector selection for cDNA libraries. It is anticipated that the readerwill then be able to
use his/her knowledge to evaluate the advantages of new systems.
Some of the factors that govern vector/host selection for cDNA library
construction are similar to those for genomic libraries. High transfection/
transformation efficiencies, stable propagation and selectable markers are
desirable. Unlike genomic libraries, the size ofthe insert is less often a problern since most fulllength cDNAs can be easily cloned into either plasmid
or lambda bacteriophage, and virtually all cDNA libraries are constructed
using one of these vectors. Historically, bacteriophage were preferred because packaging and transfection efficiencies far exceeded plasmid transformation rates and yielded a higher number of primary recombinants per
microgram of starting cDNA. Technical improvements (electroporation
and transformation of plasmids into high-efficiency competent cells)
have since made this difference negligible, and other features are now
used to select an appropriate system. Bacteriophage libraries are easier
to screen and are more likely to include Ionger gene sequences. Furthermore, one can use a modified bacteriophage which requires a minimum
insert size that is greater than the average transcript size ( eg. > Skb) to select
for Ionger cDNAs. On the other hand, plasmid libraries have some other
advantages. They are easier to amplify in a way that maintains the relative
proportion of clones within them. Plasmid DNA also is more easily prepared and transfected into eukaryotic cells for expression studies.
The most important factor that determines selection of a vector is how
the library is to be screened. As discussed previously, clones encoding genes
of interest can be identified in cDNA libraries using standard hybridization
protocols. A major advantage of cDNA libraries isthat one requires neither
the gene sequence nor a sub-fragment to isolate its fulllength clone. Because
cDNA clones are derived from mRNA, they potentially encode the protein
products of the genes from which they were derived. This feature allows an
investigator to identify genes on the basis of functional properties rather
than sequence. Antibodies, ligands, and functional attributes such as transport activity or ability to rescue function become additional tools that are
useful in identifiying clones derived from genes of interest. A yeast-based
technique, known as the two-hybrid system, has been developed to screen
for genes which encode proteins capable of interacting with a protein of
interest (bait). The assay is based on the abilitytoseparate eukaryotic tran-