48 Part I: Principles
blastocysts and grown in culture. The cultured cells
are then injected into the inner cell mass of an em-
bryo in the blastocyst stage, which is then implanted
into the foster mother, resulting in the production of
a chimeric animal (Hochedlinger and Jaenisch
2003). It is important to remember that these meth-
ods do not create new species; they only offer tools
for producing new strains of animals that carry nov-
el genetic information. Some examples of genetical-
ly engineered animals include transgenic cows that
produce milk with improved composition and trans-
genic swine that produce meat with lower fat con-
tent. The main goal of livestock genetic engineering
programs is to increase production efficiency while
delivering healthier animal food products.
MODIFIEDMILK INTRANSGENICDAIRY
CATTLE
Bovine milk has been described as an almost perfect
food because it is a rich source of vitamins, calcium,
and essential amino acids (Karatzas and Turner
1997). Some of the vitamins found in milk include
vitamins A, B, C, and D. Milk has greater calcium
content than any other food source, and daily con-
sumption of two servings of milk or other dairy
products supplies all the calcium requirements of an
adult person (Rinzler et al. 1999). Caseins represent
about 80% of the total milk protein and have high
nutritional value and functional properties (Brophy
et al. 2003). The caseins have a strong affinity for
cations such as calcium, magnesium, iron, and zinc.
There are four types of naturally occurring caseins
in milk: S1, S2, , and (Brophy et al. 2003).
They are clumped in large micelles, which deter-
mine the physicochemical properties of milk. Even
small variations in the ratio of the different caseins
influence micelle structure, which in turn can
change the milk’s functional properties. The amount
of caseins in milk is an important factor for cheese
manufacturing, since greater casein content results
in greater cheese yield and improved nutritional
quality (McMahon and Brown 1984). It has been
estimated that enhancing the casein content in milk
by 20% would result in an increase in cheese pro-
duction, generating an additional $190 million/year
for the dairy industry (Wall et al. 1997). Dairy cattle
have only one copy of the genes that encode
(s1/s2), , and -casein proteins, and out of the four
caseins, and are the most important (Bawden et
al. 1994). Increased milk -casein content reduces
the size of the micelle, resulting in improved heat
stability. -caseins are highly phosphorylated and
bind to calcium phosphate, thus influencing milk
calcium levels (Dalgleish et al. 1989, Jimenez Flores
and Richardson 1988).
Research on modification of milk composition to
improve nutritional or functional properties has
been mostly done in transgenic mice. Mice are good
models for the study of protein expression in mam-
mary glands, but they do not always reflect the same
protein expression levels as ruminants (Colman
1996). Brophy et al. (2003), using nuclear transfer
technology, produced transgenic cows carrying extra
copies of the genes CSN2and CSN3, which encode
bovine - and -caseins, respectively. Genomic
clones containingCSN2andCSN3were isolated
from a bovine genomic library. Previous studies con-
ducted with mice revealed thatCSN3had very low
expression levels (Persuy et al. 1995). In order to
enhance expression ofCSN3, the researchers created
aCSN2/3fusion construct, in which theCSN3gene
was fused with the CSN2 promoter. TheCSN2
genomic clone and theCSN2/3fusion construct
were co-transfected into bovine fetal fibroblast
(BFF) cells, where the two genes showed coordinat-
ed expression. The transgenic cells became the
donor cells in the process of nuclear transfer, gener-
ating nine fully healthy and functional cows. Over-
expression ofCSN2andCSN2/3in the transgenic
cows resulted in an 8–20% increase in-casein and
a 100% increase in-casein levels (Brophy et al.
2003).INCREASEDMUSCLEGROWTH INCATTLEMyostatin, also known as growth and differentiation
factor 8 (GDF-8), is a member of the transforming
growth factor (TGF-) family, which is responsi-
ble for negative regulation of skeletal muscle mass
in mice, cattle, and possibly other vertebrates. Myo-
statin is expressed in embryo myoblasts and devel-
oping adult skeletal muscle; it is produced as a 375-
amino-acid precursor molecule that is further
processed by enzymatic cleavage of the N-terminus
prodomain segment. The remaining C-terminus
109-amino-acid segment is the myostatin protein
(Gleizes et al. 1997). The processed protein forms
dimers that are biologically active. McPherron and
Lee (1997), through alignment of myostatin amino
acid sequences from baboon, bovine, chicken, hu-
man, murine, ovine, porcine, rat, turkey, and zebra-