chromosome and paste them to a backing sheet in an
Metaphase cells are required to prepare a standard
karyotype, and virtually any population of dividing cells
could be used. Blood is easily the most frequently
sampled tissue, but at times, karyotypes are prepared from
cultured skin fibroblasts or bone marrow cells. None of the
leukocytes in blood normally divide, but lymphocytes can
readily be induced to proliferate, providing a very
accessible source of metaphase cells.
There are many protocols for preparing a karyotype
from peripheral blood lymphocytes, but a rather
standard series of steps is involved:
• A sample of blood is drawn and coagulation
prevented by addition of heparin.
• Mononuclear cells are purified from the blood
by centrifugation through a dense medium that
allows red cells and granulocytes to pellet, but
retards the mononuclear cells (lymphocytes and
monocytes).
• The mononuclear cells are cultured for 3-4 days
in the presence of a mitogen like
phytohemagglutinin, which stimulates the
lymphocytes to proliferate madly.
• At the end of the culture period, when there is a
large population of dividing cells, the culture is
treated with a drug such as colcemid, which
disrupts mitotic spindles and prevents
completion of mitosis. This greatly enriches the
population of metaphase cells.
• The lymphocytes are harvested and treated
briefly with a hypotonic solution. This makes
the nuclei swell osmotically and greatly aids in
getting preparations in which the chromosomes
don't lie on top of one another.
• The swollen cells are fixed, dropped onto a
microscope slide and dried.
• Slides are stained after treatment to induce a
banding pattern as described above.
Once stained slides are prepared, they are scanned to
identify "good" chromosome spreads (i.e. the chromosomes
are not too long or too compact and are not overlapping),
which are photographed. The photos then are given to
kindergarten children, who cut out the images of each
karyotype before chromosomes are secured in
orderly manner. Alternatively, a digital image of the
chromosomes can be cut and pasted using a computer. If
standard staining was used, the orderly arrangement is
limited to grouping like-sized chromosomes together in
pairs, whereas if the chromosomes were banded, they can
be unambiguously paired and numbered.
The image below shows chromosomes as they are seen on
the slide (left panel) and after arrangment (right panel).
Procedure:
1. Count the number of chromosomes. Solid
stained chromosomes or chromosomes treated
with a trypsin and giemsa stain can be counted
at the microscope with a 100X magnification.
However, for analysis such as identification of
marker chromosomes or determination of the
number of copies of individual chomosomes, it
is usually necessary to photograph and print the
chromosome spreads. Refer to procedures in
this manual for black and white photography
and film development. Two prints should be
made of each spread. One will be cut for
karyotyping; the uncut print serves as a
reference if questions arise about the
interpretation of a certain chromosome.
2. Cut out each individual chromosome and
arrange on a karyotype sheet. Chromosomes are
ordered by their length, the position of the
centromere, the position of the chromosome
bands, and the relative band sizes and
distributions. Refer to page 10 of the 1985 ISCN
for the human karyotype.
3. In the construction of the karyotype the
autosomes are numbered 1 to 22, in descending
order of length. The sex chromosomes are
referred to as X and Y. The symbols p and q are
used to designate, respectively, the short and
long arms of each chromosome. There are 7
groups identified in the karyotype and data
pertaining to each group can be found on page 7
of the 1985 ISCN.
4. Secure chromosomes in place with glue. Pair the
chromosomes closely together and align the
centromeres (for easier band comparison and
checking for structural chromosome
aberrations). If possible, have a second
technologist check the interpretation of the
place.
5. A description of the karyotype should be
recorded on the karyotype sheet. First record the
number of chromosomes, including the sex
chromosomes, followed by a comma (,). The
sex chromosome constitution is given next. Any
structural rearrangements and additional or
missing chromosomes are listed next. Refer to
ISCN 1985, table 3 on page 20, for
nomenclature symbols and additional
information. Other information such as the cell
line number, the date karyotype was prepared,
the specimen type, and the technologist also
should be recorded on the
Disacovering the Cut-and-Paste Enzymes
Recombinant plas
The first major step forward in the
ability to chemically modify genes occurred when
American biologist Martin Gellert and his colleagues from
the National Institutes of Health purified and characterized
an enzyme in Escherichia coli responsible for the actual
joining, or recombining, of separate pieces of DNA
(Zimmerman et al., 1967). They called their find "DNA-
joining enzyme," and this enzyme is now known as DNA
ligase. All living cells use some version of DNA ligase to
"glue together" short strands of DNA during replication.
Using E. coli extract, the researchers next showed that only
in the presence of ligase was it possible to repair single-
stranded breaks in λ phage DNA. (Discovered in 1950 by
American microbiologist Esther Lederberg, λ phage is a
virus particle that infects E. coli.) More specifically, they
showed that the enzyme was able to form a 3'-5'-
phosphodiester bond between the 5'-phosphate end of the
last nucleotide on one DNA fragment and the 3'-OH end of
the last nucleotide on an adjacent fragment. The
identification of DNA ligase was the first of several key
steps that would eventually empower scientists to attempt
their own recombination experiments—experiments that
involved not just recombining the DNA of a single
individual, but recombining DNA from different
individuals, including different species.
A second major step forward in gene modification was the
discovery of restriction enzymes, which cleave DNA at
specific sequences. These enzymes were discovered at
approximately the same time as the first DNA ligases by
Swiss biologist Werner Arber and his colleagues while they
were investigating a phenomenon called host-controlled
restriction of bacteriophages. Bacteriophages are viruses
that invade and often destroy their bacterial host cells; host-
controlled restriction refers to the defense mechanisms that
bacterial cells have evolved to deal with these invading
viruses. Arber's team discovered that one such mechanism
is enzymatic activity provided by the host cell. The team
named the responsible enzymes "restriction enzymes"
because of the way they restrict the growth of
bacteriophages. These scientists were also the first to
demonstrate that restriction enzymes damage invading
bacteriophages by cleaving the phage DNA at very specific
nucleotide sequences (now known as restriction sites). The
identification and characterization of restriction enzymes
gave biologists the means to cut specific pieces of DNA
required (or desired) for subsequent recombination.
Inserting Foreign DNA into a New Host Cell
Although Griffith and Avery had had demonstrated the
ability to transfer foreign genetic material into cells decades
earlier, this "transformation" was very inefficient, and it
involved "natural" rather than manipulated DNA. Only in
the 1970s did scientists begin to use vectors to efficiently
transfer genes into bacterial cells. The first such vectors
were plasmids, or small DNA molecules that live naturally
inside bacterial cells and replicate separately from a
bacterium's chromosomal DNA.
Plasmids' utility as a DNA shuttle, or vector, was
discovered by Stanford University biochemist Stanley
Cohen. Scientists had already established that some
bacteria had what were known as antibiotic resistance
factors, or R factor-plasmids that replicated independently
inside the bacterial cell. But scientists knew little about
how the different R factor genes functioned. Cohen thought
that if there were an experimental system for transforming
host bacterial cells with these R-factor DNA molecules, he
and other researchers might be able to better understand R-
factor biology and figure out exactly what it was about
these plasmids that made bacteria antibiotic-resistant. He
and his colleagues developed that system by demonstrating
that calcium chloride-treated E. coli can be genetically
transformed into antibiotic-resistant cells by the addition of
purified plasmid DNA (in this case, purified R-factor
DNA) to the bacteria during transformation (Cohen et al.,
1972).
Recombinant Plasmids in Bacteria
The following year, Stanley Cohen and his colleagues were
also the first to construct a novel plasmid DNA from two
separate plasmid species which, when introduced into E.
coli, possessed all the nucleotide base sequences and
functions of both parent plasmids. Cohen's team used
restriction endonuclease enzymes to cleave the double-
stranded DNA molecules of the two parent plasmids. The
team next used DNA ligase to rejoin, or recombine, the
DNA fragments from the two different plasmids (Figure 2).
Finally, they introduced the newly recombined plasmid
DNA into E. coli. The researchers were able to join two
DNA fragments from completely different plasmids
because, as they explained, "the nucleotide sequences
cleaved are unique and self-complementary so that DNA
fragments produced by one of these enzymes can associate
by hydrogen-bonding with other fragments produced by the
same enzyme" (Cohen et al., 1973).
Phage ? is an eff
The same could be said of any DNA
—not just plasmids—from two different species. This
universality—the capacity to mix and match DNA from
different species, because DNA has the same structure and
function in all species and because restriction and ligase
enzymes cut and paste the same ways in different genomes
—makes recombinant DNA biology possible.
Today, the E. coli λ bacteriophage is one of the most
widely used vectors used to carry recombinant DNA into
bacterial cells. This virus makes an excellent vector
because about one-third of its genome is considered
nonessential, meaning that it can be removed and replaced
by foreign DNA (i.e., the DNA being inserted). As
illustrated in Figure 3, the nonessential genes are removed
by restriction enzymes (the specific restriction enzyme
EcoRI is shown in the figure), the foreign DNA inserted in
their place, and then the final recombinant DNA molecule
is packaged into the virus's protein coat and prepped for
introduction into its host cell.
Vectors Used in Mammalian Cells
A fourth major step forward in the field of recombinant
DNA technology was the discovery of a vector for
efficiently introducing genes into mammalian cells.
Specifically, researchers learned that recombinant DNA
could be introduced into the SV40 virus, a pathogen that
infects both monkeys and humans. Indeed, in 1972,
Stanford University researcher Paul Berg and his
colleagues integrated segments of λ phage DNA, as well as
a segment of E. coli DNA containing the galactose operon,
into the SV40 genome. (The E. coli galactose operon is a
cluster of genes that plays a role in galactose sugar
metabolism.) The significance of their achievement was its
demonstration that recombinant DNA technologies could
be applied to essentially any DNA sequences, no matter
how distantly related their species of origin. In their words,
these researchers "developed biochemical techniques that
are generally applicable for joining covalently any two
DNA molecules" (Jackson et al., 1972). While the
scientists didn't actually introduce foreign DNA into a
mammalian cell in this experiment, they provided (proved)
the means to do so.
To obtain rDNA steps involved are:
a) The DNA fragment containing the gene sequence to be
cloned (also known as ('insert') is isolated.
b) Insertion of these DNA fragments into a host cell using a
"vector" (carrier DNA molecule).
c) The rDNA molecules are generated when the vector self
replicates in the host cell.
d) Transfer of the rDNA molecules into an appropriate host
cell.
e) Selection of the host cells carrying the rDNA molecule
using a marker.
f) Replication of the cells carrying rDNA molecules to get a
genetically identical cells population or clone.
Enzymes used in Recombinant DNA technology
Restriction Enzymes
For cloning of DNA, the DNA is cut at specific sites, which
are recognized and cleaved by specific enzymes. These
enzymes are known as restriction enzymes. These
restriction enzymes recognize short sequences of double
stranded DNA as targets for cleavage. Different enzymes
recognize different but specific sequences, each ranging
from 4-8 base pairs. The enzymes are named by a three
letter (or four letter) abbreviation that identifies their origin
e.g. AluI is derived from Arthrobacter luteus, EcoRI is
derived from E.Coli, HpaI is derived from Haemophilus
parainfluenzae
Besides cleavage, modification in the form of methylation
is also brought about by some enzymes called modification
enzymes sometimes also called “methylases”. Different
species of bacteria contain their own sets of restriction
enzymes and corresponding methylases. Depending on the
different modes of action, the restriction enzymes have
been classified into three main classes- type I, type II, type
III. Out of these, type II restriction enzymes are used in
recombinant technology as they can be used in vitro to
recognize and cleave with in specific DNA sequences
usually consisting of 4-8 nucleotides.
DNA ligases
These enzymes form phosphodiester bonds between the
adjacent molecules and, covalently links two individual
fragments of double stranded DNA. The most commonly
used enzyme for cloning experiment is T4 DNA ligase.
Alkaline phosphatase
The enzyme alkaline phosphatase (AP) removes the
phosphate group from the 5’ end of a DNA molecule
leaving a free 5’ hydroxyl group hence it is used to prevent
unwanted self-ligation of vector DNA molecules in cloning
procedures. This enzyme is isolated from bacteria (BAP) or
calf intestine (CAP).
Cloning vectors for Recombinant DNA
Vectors are the vehicles used to carry a foreign DNA
sequence into a given host cell. A vector should have a)
origin of replication, b) a selectable marker to select the
host cells containing the vector from among those which do
not have the vector, c) one unique restriction endonuclease
recognition site to enable foreign DNA to be inserted into
the vector in order to generate a recombinant DNA
molecule and, d) it should be relatively small in size.
Plasmids as vectors
Plasmids are defined as autonomous elements, whose
genomes exist in the cell as extrachromosomal units. They
are self replicating, circular duplex DNA molecules present
in number of copies in a bacterial cell, yeast cell or in
organelles in eukaryotic cells. These naturally occurring
plasmids have been modified to serve as vectors in the
laboratory.
pBR322 vectors
One of the most commonly used cloning vector in gene
cloning procedures is pBR322 (named after Bolivar and
Rodriguez who constructed this) derived from E. coli
plasmid ColE1. It is 4,362 bp DNA with genes for
resistance against two antibiotics- tetracycline and
ampicillin. It was constructed after several alterations and
modifications in earlier cloning vectors.
pUC vectors
The plasmids belonging to pUC series of vectors are 2,700
bp long with ampicillin resistance gene, the origin of
replication derived from pBR322 and the lac Z gene
derived from E.coli. When the DNA fragments are cloned
in this region of pUC, the lac gene is inactivated.
Yeast plasmid vectors
Special vectors used to introduce DNA segments in yeast
cells or a eukaryotic cell is being used to develop
genetically engineered yeast cells. E.g. YIp or yeast
integrative plasmids which allows transformation by
crossing over and have no origin of replication. YEp or
yeast episomal plasmids carry 2 micron DNA sequence
including the origin of replication and rep gene. These
vectors have been widely used to study yeast genome.
Shuttle vectors
The vectors containing two types of origin of replication
which helps them to exist in both eukaryotic cell as well as
E.coli are known as shuttle vectors. E.g.Yep vector.
Retriever vectors
A class of vectors which are used to retrieve specific genes
from the normal chromosome of an organism like yeast
through recombination. They are very useful in isolation of
genes from yeast for molecular experiments like
sequencing.
Vectors based on bacteriophages
Bacteriophages are viruses that infect bacterial cells by
injecting their DNA into these cells. They are used as
vectors because they have a linear DNA molecule, which
generate two fragments after breakage. These are later
joined with foreign DNA to generate chimeric phage
particle. The injected DNA is selectively replicated and
expressed in the host cell resulting in a number of phages
which burst out of the cell (lytic pathway) and further infect
the neighbouring cells. E.g. M13 , Lambda ( )
Cosmids as vectors
Cosmids are plasmid particles into which specific DNA
sequences like cos sites of phage lambda are inserted and
they are constructed by combining certain feature of
plasmids and the ‘cos’ sites of phage lambda. The
advantage of the using cosmids for cloning is that its
efficiency is high to produce a complete genomic library of
10(6)-10(7) clones from only 1 microgrm of insert DNA.
Phagemids as vectors
Phagemids are prepared artificially by combining features
of phages with plasmids. E.g. pBluescript II KS is derived
from pUC19 and is 2961 bp long.
BAC Vectors
BACs or Bacterial Artificial Chromosomes are vectors
based on the natural, extra-chromosomal plasmid of E.coli-
the fertility or F-plasmid, and are being used in genome
sequencing projects. A BAC vector contains genes for
replication and maintenance of the F-factor, a selectable
marker and cloning sites and can accommodate up to 300-
350 kb of foreign DNA.
Plant and animal viruses as vectors
A number of plant and animal viruses can also be used as
vectors for introducing foreign genes into cells and/or for
gene amplification in host cells. Plant viruses like Gemini
viruses, cauliflower mosaic virus or CaMV and tobacco
mosaic virus /TMV) are three group of viruses that have
been used as vectors for cloning of DNA segments. CaMV
has a double stranded DNA molecule, 8kbp in size which
infects the members of Cruciferae family. Geminiviruses
are a group of single stranded DNA plant viruses infecting
cassava, maize and other cereals.
Animal virus vectors also deliver the foreign genes into the
cultured cells which get integrated into the host genome.
The expression of foreign genes can also be amplified
using the promoters of the virus gene. The cloned genes
can be used in gene therapy, for the synthesis of important
proteins etc. A vector based on Simian Virus 40 (SV 40)
was used in the first cloning experiment involving
mammalian cells in 1979. Retroviruses like murine and
avian retroviruses are very useful vectors as they are
capable of infecting a large variety of cell types and can
clone large genes. Herpes virus is a non-integrating large
sized virus (150 kb) which is another useful vector which
can be used for expression of large intact genes.
Artificial chromosome (YAC and MAC) vectors
YACs or Yeast Artificial Chromosomes are used as vectors
to clone DNA fragments of more than 1Mb in size. These
long molecules of DNA can be cloned in yeast by ligating
them to vector sequences that allow their propagation as
linear artificial chromosomes. These long DNA molecules
can be generated and allow construction of comprehensive
libraries in microbial hosts. A lot of work is going on to
create mammalian artificial chromosomes (MACs)
following the isolation of mammalian telomeres and
centromere. However YACs have a low cloning efficiency
(1000 clones/microgm) DNA as against 106-107
clones/microgm DNA for cosmids) and also it is difficult to
recover large amount of pure insert DNA from individual
clones.
Transgenic Animals
Another use of rDNA technology is to add an outside
gene to the DNA of animals, creating a transgenic animal.
These genes are added to the animal before it is born.
Genes can be inserted into the animal to alter its protein
content--for example, to produce a cow with low-lactose
milk. Transgenic pigs might have organs that can be used
for human transplantation. Creating disease-resistant
animals is another possibility with rDNA technology. .
I. DEFINITION OF RECOMBINANT DNA:
1. DNA molecules contructed outside of living cells by
joining natural or synthetic DNA segments to DNA
molecules that can replicate in a living cell
2. DNA molecules resulting from replication of a molecule
described in 1. above.
II. GOALS OF RECOMBINANT DNA
TECHNOLOGY
A. To ISOLATE and CHARACTERIZE a gene
B. To MAKE DESIRED ALTERATIONS in one or more
isolated genes
C. To RETURN ALTERED GENES TO LIVING
CELLS (or to multicellular animals or plants) to
EXAMINE OR UTILIZE THEIR EXPRESSION
III. BASIC TOOLS OF RECOMBINANT DNA
TECHNOLOGY:
A. NUCLEIC ACID ENZYMES: DNA polymerases,
reverse transcriptase, DNA ligase, RESTRICTION
ENDONUCLEASES, Table 3.2
B. BACTERIAL GENETICS: PLASMIDS,
BACTERIOPHAGES (bacterial viruses)
a. Bacteriophages replicate via the LYTIC PHAGE
CYCLE: the phage genome is injected into the cell, phage
genes are expressed and phage proteins and DNA are made,
progeny phage are packaged, and the cell is lysed, Fig.
5.37. Two genetically different phage that infect the same
host cell may recombine during the lytic cycle,
b. Some PHAGE can also replicate via the LYSOGENIC
CYCLE. The phage genome is integrated into the host
chromosome and becomes copied into chromosomes of all
daughter bacteria like any gene. This "PROPHAGE" can
be induced to enter the lytic cycle and kill its host by a
variety of stresses like UV light. Fig. 5.37
c. Plasmids: Circular DNAs that replicate
autonomously; (Fig. 3.22)
C. ANIMAL VIRUSES AND ANIMAL CELL
CULTURE TECHNIQUES
V. VECTORS: A VECTOR IS A DNA MOLECULE
INTO WHICH FOREIGN DNA MAY BE INSERTED
WHICH CAN THEN REPLICATE IN AN
APPROPRIATE CELL.
A. Vectors must have one or more ORIGIN OF
REPLICATION, and
B. One or more SITE into which the recombinant DNA
can be inserted.
C. They often have convenient means by which cells with
vectors can be SELECTED from those without: e.g.,
DRUG RESISTANCE GENES, Fig. 3.22.
D. Common vectors include PLASMIDS, VIRAL
GENOMES, and (primarily in yeast) "ARTIFICIAL
CHROMOSOMES". A SHUTTLE VECTOR is one that
can replicate in 2 or more types of cell (e.g., E. coli and
yeast, Fig. 3.34A).
V. JOINING DNA FRAGMENTS: The most common
mechanism of JOINING the TARGET DNA into the
vector DNA is with COHESIVE RESTRICTION
ENZYME ENDS ("sticky ends") sealed by DNA
LIGASE, Fig. 3.19.
VI. TARGET DNA: By "target DNA" we mean the DNA
or gene that one wishes to isolate and use as described
above. As specified in the definition of recombinant DNA,
the target DNA must be joined to another DNA that can
replicate (the vector). Target DNA can be of several forms
including:
A. CHROMOSOMAL DNA ("GENOMIC DNA")
isolated directly from cells or a tissue
B. COMPLEMENTARY DNA (cDNA) made in vitro (in
the test tube) using the enzyme reverse transcriptase and
isolated mRNA as template,
C. SYNTHETIC DNA (rarely), made by machines that
rely on organic chemistry
VII. RECOMBINANT DNA CLONING
A. "CLONING" is the isolation and growth of a single,
genetically uniform cell and its offspring. Recombinant
DNA cloning is the isolation of a cell line which contains
(and replicates) a single, unique recombinant DNA from a
pool (LIBRARY) of many different recombinant DNA’s,
Fig. 3.33.
B. To clone (as with all isolation or purification
techniques), you need two things: a SEPARATION
METHOD and an ASSAY. The most common assay for a
specific DNA is NUCLEIC ACID HYBRIDIZATION,
Fig. 3.28. SEPARATION METHOD: With recombinant
DNA technology one can use bacteria or bacteriophage to
readily separate thousands or even millions of unique
colonies or plaques in a clone bank or library.
Cloning DNA from an animal or any other organism is
taking a piece of the purified DNA from that organism
and putting it into a bacterium (usually). One then
isolates a single bacterial colony (or CLONE) which can
be used to make millions and millions of copies of the
single DNA fragment you wish to study. The common
use of the term "cloning" for recombinant DNA cloning
like that just described is an unfortunate coincidence of the
way these bacteria were first made. Note that recombinant
DNA cloning involves isolated, purified DNA; one works
with only a small portion or fragment of the animal's
genome; and that only new bacterial colonies are generated.
In other words, if someone sent you DNA from a blue
whale, you could "clone" blue whale DNA even though
you've never seen a whale and have no intention ever to see
one in your life.
TRANSGENIC ANIMALS AND PLANTS,
A. GERMLINE: TRANSGENE is integrated into the
chromosome of a germ line cell and can be passed on to
offspring as a Mendelian trait.
B. SOMATIC: TRANSGENE is integrated into
chromosome(s) in some cells or tissues, but cannot be
inherited by offspring (e.g., Gene Therapy) .
C. Among the most useful transgenics are TRANSGENIC
MICE, which can be made by growing EMBRYONIC
STEM (ES) CELLS in culture dishes (Fig. 3.38),
transfecting them with DNA by standard procedures and
then INJECTING THE ALTERED ES CELLS into a
blastocyst stage (early) embryo. The product offspring is
genetically CHIMERIC. If the chimerism extends to the
germline, some of the offspring of the chimeric mouse will
be GERMLINE TRANSGENICS. Transgenic mice can
also be made by MICROINJECTING PURE DNA
INTO THE PRONUCLEUS (Fig. 3.37) of a 1 cell
embryo and reimplantation of the injected embryo. Some of
these can survive and produce complete or chimeric
transgenics. Other transgenic vertebrate species similar
have been made, but with less routine success to date
Animal Husbandry
Neither the use of animal vaccines nor adding bovine
growth hormones to cows to dramatically increase milk
production can match the real excitement in animal
husbandry: transgenic animals and clones.
Transgenic animals model advancements in DNA
technology in their development. The mechanism for
creating one can be described in three steps:
1. Healthy egg cells are removed from a female of
the host animal and fertilized in the laboratory.
2. The desired gene from another species is
identified, isolated, and cloned.
3. The cloned genes are injected directly into the
eggs, which are then surgically implanted in the
host female, where the embryo undergoes a
normal development process.
Transgenics
Cloning in Animals.
Animals have two different classes of cells: germ line
and somatic.
Only alterations in the germ cells can be transmitted to
future generations. However, some forms of somatic
cell gene therapy can be useful in treating patients. For
example, people with cystic fibrosis can receive the
cloned CFTR gene in a nasal spray, which then infects
the lining of their lungs and improves lung function.
The infected (transformed) epithelial cells eventually
are lost, however, during normal processes of tissue
growth, so the treatment needs to be repeated.
Germ-line cloning. There are two basic ways to clone a
gene in the mammalian germ line.

• Inject a DNA fragment containing your gene

into the nucleus of a fertilized egg (germ-line
gene cloning) or of body tissues in a mature
host (somatic gene cloning). The DNA gets
taken up at random somewhere in one of the
chromosomes.

An example of germ-line cloning is the

injection of angiopoietin cDNA into the
fertilized mouse egg.
(An example of somatic cloning is gene
therapy for cystic fibrosis: inhale a vector
containing the CF gene, which gets
incorporated into the DNA of cells lining the
lungs. Somatic cloned genes are not inherited
by offspring.)

• Put a transgene containing positive and

negative selective markers into an embryonic
stem cell tissue culture. The ES cells take up
the gene by homologous recombination,
replacing the host allele. The ES cells now
are injected into a blastula of an unrelated
host, and some of the next generation
progeny arise from the ES cells. To learn
more about this, read Capecchi's article on
Targeted Transgenes, on reserve.