Cloning Genes for Chapter

e7
Analysis

Genes Are Cloned Onto

Vectors...........................................e83

Genes Are Cloned Onto Vectors Detecting Cloned DNA................e84

Usually when one hears the term “cloning,” thoughts of Dolly the sheep Moving Genes Between
and CC the cloned cat surface. In fact, cloning genes is a much simpler Different Organisms....................e85
process and is quite common. Cloning genes involves the amplification
Dealing with Very Large DNA
of a target gene from one organism using PCR (Ch. 6) and the insertion
of that PCR product into a small, circular piece of DNA called a clon- Segments......................................e85
ing vector. The cloned target DNA is then used in a variety of processes,
Cloning by Recombineering.......e86
including sequencing, production of libraries, controlled expression of
the gene product, and many other applications discussed in this chapter. Gene Libraries..............................e86
The Key Concepts for this chapter are discussed below.
Chromosome Walking and
l Taking a gene from one organism and expressing it in a differ- Subtractive Hybridization...........e88
ent one requires the use of cloning vectors, which are relatively
Expression Vectors Allow
small rings of DNA that move from cell to cell.
High Level Protein Production...e88
Cloning vectors are usually modified plasmids, which are small,
circular pieces of DNA that can replicate themselves independently of
the host cell’s chromosome. They are found in many organisms, includ-
ing bacteria and yeast. Cloning vectors have special features that ena-
ble them to be easily manipulated by introducing foreign genes into
them. They are usually small and easy to manage. Also, they are eas-
ily transferred from one cell to another and subsequent generation of
multiple copies of the vector can then be easily isolated.
Furthermore, vectors have been genetically enhanced for clon-
ing and expression of the target sequence by addition of several fea-
tures. Often, extra genes are added to the vectors to provide a means
for a researcher to detect their presence in a population of cells.
Additionally, to facilitate cloning, a multiple cloning site is added. This
site contains many restriction sites that can be used to insert the target
sequence. Some vectors even contain genes that, when disrupted by the
insertion of cloned DNA, allows detection of the presence of an insert.

l ColE1 plasmids of E. coli are the most common and widely

used vectors. The original plasmids have had their genes for
colicin production removed and replaced with a gene for anti-
biotic resistance so that a bacterium harboring this plasmid will

Molecular Biology, Second Edition Study Guide.

© 2013 Elsevier Inc.
Academic Cell is an imprint of Elsevier Inc. e83
e84 CHAPTER SEVEN • Cloning Genes for Analysis

become resistant to that antibiotic. This phenotype distinguishes bacteria con-

taining the plasmid from those without the plasmid.

The ColE1 plasmid from E. coli is commonly manipulated for use in molecular
biology. The copy number of a plasmid determines how many copies of that particular
vector can reside within a host cell. The original ColE1 plasmid can exist as 40 copies
in a host cell, which helps fulfill the requirement of cloning vectors to be easily gener-
ated and isolated.
The most common antibiotic resistance gene inserted into the ColE1 plasmid in
place of the colicin is for the antibiotic ampicillin. This encodes β-lactamase, which is
an enzyme that degrades penicillin, ampicillin, and related antibiotics. The presence of
the antibiotic resistance gene is a powerful tool for selecting for those cells that con-
tain the vector, as they will be resistant to the antibiotic and will be the only cells to
survive in its presence.
l DNA can be inserted into a vector by digesting the piece of DNA and vec-
tor with the same restriction enzyme and ligating the two. Polylinkers or multi-
ple cloning sites in a vector have a series of unique restriction enzyme sites to
use. Alternatively, PCR amplified DNA inserts have a single adenine extension
onto the 3´ end of each strand that can be cloned into a TA vector that has a
single thymine overhang.

Digesting a vector and target sequence ends with the same restriction enzyme
will yield complementary sticky ends for both vector and sequence. The two pieces of
DNA are ligated using DNA ligase. One feature of many cloning vectors is their engi-
neered multiple cloning site, which contains a variety of restriction sites. A researcher
can choose a restriction site in the multiple cloning site that is not present in the
target sequence or anywhere else on the vector. Otherwise, cutting the DNA with a
restriction enzyme that recognizes a sequence in the middle of the target DNA will
produce unwanted fragmenting.
Some cloning vectors take advantage of the Taq polymerase (DNA polymerase
commonly used in PCR, Ch. 6) property of adding a single adenine onto the 3´ end of
a PCR-amplified strand. This terminal transferase activity is exploited by TA vectors,
which contain single thymine overhangs to which the single adenine can bind.

Detecting Cloned DNA

l Insertional inactivation is a method to detect the presence of an insert in a vec-
tor, whereby the DNA insert is cloned so that it disrupts a gene for antibiotic
resistance. The bacterium harboring the vector with insert is no longer resist-
ant to that antibiotic and can be discerned from those bacteria harboring the
vector without an insert.
l Beta-galactosidase is a common reporter gene used to detect the presence

of an insert in a vector. When the vector has no insert, the alpha fragment of
β-galactosidase is made and combines with the other half of the enzyme. The
active enzyme then converts the X-gal into a precursor that reacts with oxygen
to create a blue dye. When the insert disrupts lacZ, no alpha fragment is made,
and the bacterial colony remains white on X-gal plates.

Several methods are employed to detect the presence of an insert within a vector.
In one case, two antibiotic genes are present on the vector. One is used for the selec-
tion of the plasmid itself and the other is used for detection of the insert. The target
DNA is cloned in the middle of one of the antibiotic resistance genes, which disrupts
the gene. Now, the host cell is not resistant to the second antibiotic. The outcome is
that cells containing the vector but no insert are resistant to both antibiotics. Cells
that have acquired the vector with the insert are only resistant to one antibiotic.
Reporter genes are also used to detect the presence of inserts within a vector.
One such reporter gene, called β-galactosidase (encoded by lacZ), can convert a clear
chemical (X-gal) into a substance that turns blue in the presence of oxygen, which
 Dealing with Very Large DNA Segments e85

is called blue/white color screening. The vector is designed to contain a selective

marker, such as antibiotic resistance, and the coding sequence for only the first 146
amino acids of β-galactosidase. The multiple cloning site is within the first portion of
the lacZα gene on the vector. The remainder of the lacZ gene is contained within spe-
cial host cells. If a target DNA molecule inserts into the vector, the cells will be white
when grown in the presence of X-gal, due to the disruption of lacZα. Cells that harbor
the vector without the insert will be blue in the presence of X-gal because no disrup-
tion of lacZα occurred. Cells that do not acquire the vector, or vector plus insert, will
not grow in the presence of the antibiotic and will not be observed.

Moving Genes Between Different Organisms

l Shuttle vectors can survive in two different organisms, and include two origins
of replication, one for each organism, and two genes for selection, one for each
organism.

Researchers often wish to move vectors from one organism to another. The shuttle
vector is useful for performing such movements but must have a few features that
allow it to survive within different organisms. The shuttle vector must contain an origin
of replication for both organisms, as these are sequences that are recognized differently
by proteins from different species.
If the shuttle vector is to be used in a eukaryote such as yeast, then it must also
contain a centromere sequence so that microtubules can bind to it and it is properly
segregated during cell division.
Finally, two selective markers must be used, one for each organism. Antibiotics
are usually specific to bacterial cells, but have no effect on eukaryotic cells such as
yeast. If yeast is a desired organism for the shuttle vector, then some other selecta-
ble marker must be used. Often, strains defective in certain metabolic pathways are
used. If the yeast strain is defective in one enzyme for the manufacture of an amino
acid, then the cells are not able to grow without being given exogenous amino acid.
However, if the functional copy of the DNA for the defective enzyme is present
on the shuttle vector, then the yeast can now grow without the addition of the spe-
cific amino acid. The metabolic differences give researchers a phenotypic difference
between cells that have the vector and those that do not have it.

Dealing with Very Large DNA Segments

l The genome from lambda virus has been converted into a vector for large
DNA inserts (about 23 kb) by removing the central region of the genome,
which contains the genes for integration and recombination into the E. coli
chromosome. The large DNA insert can replace this region and can be inserted
into E. coli bacteria using in vitro packaging. Cosmid vectors are about 45 kb of
DNA flanked by the cos ends of the lambda genome. These are also inserted
into E. coli using in vitro packaging.
l Artificial chromosomes from yeast, bacteria, or P1 bacteriophage are used for

even larger DNA inserts (up to 150 kb).

Various methods also exist for cloning larger segments of DNA. The bacteri-
ophage lambda, has been altered by removing large portions of the lambda genome.
Large target DNA inserts can be placed into these regions and then inserted into
the E. coli chromosome. A technique called in vitro packaging is often employed to
accomplish this. In this technique, lambda DNA carrying the inserts is mixed with viral
proteins in a test tube. The proteins and DNA combine to form phage particles. The
lambda proteins are derived from two cultures of E. coli cells infected with lambda
mutants. Each mutant was defective in a different aspect of phage head assembly.
When brought together, the head proteins combine to generate functional phage.
Cosmid vectors are also lambda-derived vectors and can hold up to 45 kb of DNA.
e86 CHAPTER SEVEN • Cloning Genes for Analysis

For very large inserts, yeast artificial chromosomes (YACs), bacteria artificial
chromosomes (BACs), and P1 bacteriophage artificial chromosomes (PACs), are used.
YACs are able to hold 2,000 kb and contain a yeast origin of replication, along with
centromere recognition sequences, and telomere sequences present on both ends.
YACs are also engineered with selectable markers and multiple cloning sites.
BACs can hold up to 300 kb and are used to clone large segments of eukaryotic DNA
in bacteria, such as for the human genome project and other sequencing projects. BACs
are derived from the F plasmid of E. coli, which is normally involved in conjugation.
PACs are derived from the P1 bacteriophage and can carry 150 kb inserts.
Because they are phage-derived, in vitro packaging is required, similar to that used
for lambda-derived vectors.

Cloning by Recombineering
l Recombineering inserts specific pieces of DNA into a vector or artificial chro-
mosome by homologous recombination. The RED system from bacteriophage
lambda recognizes the ends of the insert with exact homology to the insertion
site on the vector, and recombines the DNA insert with the vector to make the
two pieces one.

Lambda can undergo two life cycles: lysogenic and lytic. In the lysogenic viral life
cycle, lambda can integrate its DNA into the host cell’s genome and hide from the
host cell. The integrated lambda DNA is not recognized by the host cell as foreign,
and it is replicated along with the host’s chromosome in cell division so that each of
the daughter cells contains a lambda copy.
In homologous recombination, pieces of DNA that are homologous swap places
with each other. A lambda system for recombination, called RED, is currently
exploited as the most commonly used recombineering system. Recombineering is a
term to describe genetic engineering that occurs via homologous recombination.
The RED system is used by lambda to integrate its genome into the host cell. This
system can be used to integrate any linear piece of DNA and has several advantages over
traditional cloning methods. The RED proteins do not cause spontaneous mutations
when expressed, at least over short periods of time. The homologous region only needs to
be about 45 bp for integration into the vector. The procedure is relatively quick and easy.
Small deletions, insertions, and nucleotide changes can be made with this system.

Gene Libraries
l DNA libraries are constructed by partially cutting the genome of interest with
a restriction enzyme to generate large fragments, inserting each of the frag-
ments into a vector, and then putting each vector into a bacterial cell. Each
bacterium in a library has a different part of the genome.
l DNA libraries can be screened by denaturing a labeled probe and denaturing

the vector and insert into single-stranded DNA, and then, mixing these at a
temperature that is sufficient for the probe to hybridize to the target. The lower
the temperature, the less similar the hybridization sequence will be to the probe,
and the higher the temperature, the more similar the two sequences will be.

Gene libraries contain segments of genomes cloned onto individual vectors. The
libraries are very large and contain a collection of all the genes from an organism. To
construct a library, total genomic DNA from an organism is cut with one restriction
enzyme. The resulting DNA fragments are cloned into a vector, such as ColE1, and
then transformed into a suitable host cell. Large numbers of transformants are then
kept and screened for the cloned genes. Usually this screening involves hybridization
experiments to DNA probes made from a related organism or artificially synthesized.
The hybridization specificity of the probe to the target sequence can be changed
by altering the temperature. Higher hybridization temperatures usually mean more
 Gene Libraries e87

Song H, Chung S-K, and Xu Y. (2010) Modeling disease in human ESCs
using an efficient BAC-based homologous recombination system. Cell Stem Cell
6: 80-89.
Mouse models for human genetic diseases are sometimes insuf-
ficient for some studies. This is mostly due to some differences in how the mouse
models respond to human diseases. Therefore, the need for a more comparable
model has lead researchers to utilize human embryonic stem cells (hESC). These cells
are undifferentiated and have the ability to become any cell in the body, although
they are difficult to genetically alter. Some traditional approaches have been used
with limited success. In this article, the authors describe the adaptation and optimiza-
tion of the BAC-based approach to generate hESCs that are missing ATM and p53.
ATM is a protein kinase that, when mutated, has a multisystemic effect on the
host called Ataxia-telangiectasia. This syndrome is characterized by decreased men-
tal capacity, neuronal degeneration, germ cell defects, immunodeficiency, and an
increased risk of cancer formation. p53 is a protein that regulates the cell cycle.
When p53 is mutated, the cell cycle is uncontrolled, which also can lead to tumor
formation. Mutated p53 is common in many human cancers.
The authors utilized a BAC-based vector system to generate hESC
lines missing ATM. They used a neomycin resistance cassette to screen for the
presence of the vector in the cells. BAC vectors have large recombination arms,
which increases the efficiency of homologous recombination. Using the BAC-based
approach, the authors were able to obtain an increased targeting efficiency of 21%.
One challenge with generating homozygous ATM-/- is the alteration of both ATM
alleles in the cell. To target the second allele, the authors utilized the same BAC-
based approach, but added the selection cassette for puromycin resistance, another
antibiotic. The targeting efficiency for this was 27%. The authors were successful in
generating a homozygous ATM hESC line for further analysis.
The authors also constructed a homozygous p53 hESC line using
recombineering technology for the same BAC-based system. The same sequence of
events for homozygous ATM-/- was employed. Although the targeting efficiency was
low for the first allele of p53, the second allele was successfully deleted and the
authors were able to show that their system could be used for other alleles.
The same hESC line was used for the generation of ATM-/- and p53-/-
mutants. The authors also used their BAC-based system to generate similar mutations
in other hESC lines, which indicates no relevance of cell line on their ability to generate
mutants using this system. Additionally, they were able to show that the genetically
engineered hESC behaved as expected for their respective homozygous mutations,
ATM and p53.
The use of the BAC-based system to alter large genes in human ESCs
provides a powerful tool for the study of human diseases in a more relevant model.
FOCUS ON
RELEVANT RESEARCH
Conceptual questions
1. Why was a BAC-based technology employed in the construction of the hESC lines?
2. Why did the authors not use a more traditional approach?
3. What are some advantages of using a BAC-based approach? Can you think of
any disadvantages?
Discussion points
The ability to manipulate human embryonic stem cell lines has the
potential to greatly increase our knowledge regarding normal disease progression,
drug targets, and treatments. Recently, somatic cells have been reprogrammed into
induced pluripotent stem cells (iPSC), which are stem cells that have been induced
to differentiate into specific cells. Human embroyonic stem cells have the ability to
become any type of cell in the body and are not yet differentiated into a specialized
role (i.e., nerve cells, blood cell, epithelial cell, etc.). Do you think the BAC-based
technology could be applied here as well? Can you think of any advantages of using
iPSCs over hESCs? Are there any ethical implications of using iPSCs over hESCs?

stringent conditions and only those probes that have more similiarity, although com-
plementary, to the target sequence will bind. Lowering the temperature means less
stringent conditions and more room for error between the probe and target sequence.

l Rather than screening for DNA sequences, expression of the cloned DNA
insert into protein allows the protein to be screened by antibodies.

Libraries can also be screened by expressing the cloned gene into protein and
then using antibodies that target the protein, which is called immunological screening.
One downside to this approach is that the antibody to the target protein is required.
In addition, the vector must contain an open reading frame that has both transcrip-
tional and translational start and stop sites. Otherwise, no gene expression occurs.

l Genomic DNA from eukaryotes cannot be made into an expression library

since the genes contain introns. Using cDNA circumvents this problem, and
therefore, can be used for screening by immunological methods.

Since eukaryotic DNA is often littered with intervening, non-coding sequences

called introns, cloning these genes into expression vectors is problematic. However,
total RNA from the eukaryotic cells is extracted. The mRNA is specifically targeted
and isolated by using oligo(dT) columns that bind up the mRNA only, since they are
the only type of RNA that contains poly(A) tails. The isolated mRNA, which should
e88 CHAPTER SEVEN • Cloning Genes for Analysis

already have the introns spliced out, is converted into a cDNA copy by reverse tran-
scriptase. The cDNA copy is then cloned into the expression vector, The resulting
coding sequence is much shorter than the original gene and easier to express in the
unnatural setting.

Chromosome Walking and Subtractive Hybridization

l Hybridization of one library clone to another can find overlapping regions
of DNA, and hence can be used for identifying upstream and downstream
sequences from the region of interest. This is called chromosome walking.

Chromosome walking is a useful tool to elucidate regions upstream and down-

stream of a known target sequence. In this very slow technique, the chromosome of
interest is first targeted by FISH (Ch. 5) and isolated by FACs (Ch. 5). The isolated
chromosome is then cut by a restriction enzyme and the resulting fragments are used
as probes, which will overlap. As the probes overlap, the sequence can be determined
piece by piece. The sequence both upstream and downstream of the target DNA can
be determined if walked in both directions.

l In subtractive hybridization, two different mRNA or DNA samples under two

different conditions are isolated and hybridized. Any mRNA or DNA found in
both conditions will hybridize, and any unique sequences will not. The unhybrid-
ized mRNA or DNA can then be purified and cloned into a vector for analysis.

Subtractive hybridization is used to isolate target DNAs that are missing from a
particular sample. As the key concept indicates, the mRNA or DNA from two dif-
ferent samples, each from a different condition, are isolated and hybridized to each
other. For DNA samples, the two different conditions are usually DNA from a per-
son with a genetic disease in comparison to DNA from a person without the disease.
These two conditions reveal the genetic lesion that underlies the disease. For mRNA
samples, the two conditions can also compare disease states, but these comparisons
can also compare different environmental assaults. For example, the same cells can
be tested for treatments with or without heat, with or without new candidate drugs,
or testing of potentially hazardous chemicals. Additionally, subtractive hybridization
of two mRNA samples could be from two developmental stages. Only sequences that
do not have a counterpart in the other experimental sample will be left unpaired. Any
sequence that does not hybridize is isolated from the other genetic information and
further analyzed by cloning into a vector.

Expression Vectors Allow High Level Protein Production

l Expression vectors have promoters for the DNA insert that are inducible, that
is, they are only expressed under certain conditions or with certain polymerases.

Researchers often clone genes into vectors in order to purify the protein pro-
duced by the gene. Expression vectors are engineered with special features to con-
trol expression of protein from the cloned gene. Without the control, the gene product
could potentially disrupt the host cell and even kill it. Usually an inducible promoter
is located upstream of the cloned gene. The inducible promoter is usually not active
until the researcher adds the inducer molecule to the growing cells. Some examples of
inducible promoters include the lac promoter from E. coli, which is induced by addi-
tion of the artificial inducer IPTG. The tet operon is also commonly used. The inducer
molecule for this operon is the antibiotic tetracycline.
Additionally, expression vectors often contain tags that, when expressed, produce
a peptide sequence linked to the protein of interest that is then used to isolate the
protein. One common tag includes 6X-His tags (6 histidine amino acids in a run) that
bind to divalent cations.