Shakweer et al.

Journal of Genetic Engineering

Journal of Genetic Engineering and Biotechnology (2023) 21:55
https://doi.org/10.1186/s43141-023-00502-z and Biotechnology

REVIEW Open Access

A review of transgenic animal techniques

and their applications
W. M. E. Shakweer1* , A. Y. Krivoruchko2, Sh.M. Dessouki3 and A. A. Khattab4

Abstract
Nowadays, breakthroughs in molecular biology are happening at an unprecedented rate. One of them is the ability
to engineer transgenic animals. A transgenic animal is one whose genome has been changed to carry genes from
another species or to use techniques for animal genome editing for specific traits. Animal features can be changed
by purposefully altering the gene (or genes). A mouse was the first successful transgenic animal. Then pigs, sheep,
cattle, and rabbits came a few years later. The foreign-interested genes that will be used in animal transgenic tech-
niques are prepared using a variety of methods. The produced gene of interest is placed into a variety of vectors,
including yeast artificial chromosomes, bacterial plasmids, and cosmids. Several techniques, including heat shock,
electroporation, viruses, the gene gun, microinjection, and liposomes, are used to deliver the created vector, which
includes the interesting gene, into the host cell. Transgenesis can be carried out in the gonads, sperm, fertilized eggs,
and embryos through DNA microinjection, retroviruses, stem cells, and cloning. The most effective transgenic marker
at the moment is fluorescent protein. Although transgenesis raises a number of ethical concerns, this review concen-
trates on the fundamentals of animal transgenesis and its usage in industry, medicine, and agriculture. Transgenesis
success is confirmed by the integration of an antibiotic resistance gene, western and southern blots, PCR, and ELISA. If
technology solves social and ethical problems, it will be the most promising in the future.
Keywords Transgenic animals, Vectors, DNA microinjection, Sperm-mediated gene transfer, Somatic cell nuclear
transfer, Xenotransplantation

Background and/or compositions, and increased disease resistance are

The transgenesis technique involves the introduction of for- some of the practical applications of transgenesis in animal
eign DNA sequences into the genome of transfected cells and production. Growth hormone is one of the most important
ensuring that the DNA sequences are integrated and trans- candidate genes used to produce transgenic farm animals
mitted to the offspring [1]. Greater prolificacy and reproduc- to increase their growth rate and milk production [2–4]. In
tive performance, improved feed utilization and growth rate, germ-line gene transfer, the parents’ egg and sperm cells are
improved carcass composition, improved milk production altered in order to pass the alterations on to the progeny of
the transformed species [5–7]. Nowadays, the gene con-
*Correspondence: structs have now been introduced into the majority of food
W. M. E. Shakweer animals, including cattle, sheep, goats, pigs, rabbits, chick-
[email protected]; [email protected]
1
Animal Production Department, Agricultural and Biological Research
ens, and fish [8–11]. The stable insertion of the gene into
Institute, National Research Centre, 33 El‑Buhouth Street, Dokki, the germ line has been a great technological achievement
Cairo 12622, Egypt
2
in agriculture. Animals with large transgenes are helpful for
Genetic and Biotechnology Department, All-Russian Research Institute
of Sheep and Goat Breeding, Stavropol, Russia
biotechnology and genetic research, such as the characteri-
3
Department of Animal Production, Faculty of Agriculture, Cairo zation and modulation of large single-gene and polygenic
University, 7 Gamaa Street, Giza 12613, Egypt
4
features [3, 4]. As a result, the focus of this review will be on
Genetics and Cytology Department, Biotechnology Research Institute,
National Research Centre, 33 El‑Buhouth Street, Dokki, Cairo 12622, Egypt
the most important animal transgenesis procedures.

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Shakweer et al. Journal of Genetic Engineering and Biotechnology (2023) 21:55 Page 2 of 14

Approaches to generate transgenic animals as foreign DNA vectors [13]. Various forms of viral vec-
Various methods for producing transgenic animals tors are now being used or investigated, including the
have been developed during the last few decades. Many following:
sequences have been determined as a result of gene
sequencing, bringing knowledge of promoters and genes Retroviral vectors
of relevance to many species. The advent of genom- They are RNA viruses that can generate DNA from RNA
ics, proteomics, and a new generation of reproductive using reverse-transcriptase enzymes. They can copy
biotechnologies all point to successful transgenic appli- themselves when a cell divides by integrating into the
cations in domestic animals. The procedures and meth- host DNA [5, 14, 15]. Recently, retroviral vectors were
odologies used in the creation of a transgenic animal are used to allow for the integration of a foreign gene into the
determined by the animal’s intended use. Many trans- host genome. They can carry up to 7 to 8 kb from foreign
genic animal models have been developed to research genes, but at the same time, this may not be enough for
gene function, serve as bioreactors, and serve as models long genes or structures that require extensive regulatory
for novel animal breeding techniques [1]. The primary sequences for transcription [5, 13].
approaches utilized to create transgenic animals are
shown in Fig. 1. There are three types of foreign DNA Adeno‑associated virus (AAV) vectors
transfer techniques: DNA microinjection into pronu- Adeno-associated virus (AAV) was initially detected in
clei, mass gene transfer using gametes, and somatic cell human tissues in the mid-1960s from laboratory adeno-
nuclear transfer (SCNT). virus (AdV) preparations [15, 16]. A few research groups
set out to grasp basic AAV biology out of pure scientific
Vector‑mediated gene transfer interest and without recognizing its enormous potential
The term “cloning vector” refers to a short amount of as a human gene therapy platform [16–18]. Several fun-
DNA with foreign DNA that has the capacity to repro- damental characteristics of the virus were defined during
duce itself for use in transferring or propagating in the first 15–20 years of research, including its genome
an organism. Vectors increase the probability of gene layout and composition [19], DNA replication and tran-
expression [12]. The various accessible vectors have been scription [20], infectious latency [21], and virion assem-
developed to hold DNA of various lengths. Plasmids, bly [22]. The successful cloning of the wild-type AAV2
cosmids, the P1 phage, BACs (bacterial artificial chro- sequence into plasmids, which permitted genetic studies
mosomes), and YACs (yeast artificial chromosomes) may [23], and the sequencing of the full AAV2 genome [24],
each hold 20 kilobytes (kb), 40 kb, 90 kb, 200 kb, and was made possible by these accomplishments. These
1000 kb of DNA. Viruses have the ability to deliver their early studies provided crucial information that led to the
genome into cells efficiently. Researchers were motivated development of AAV as a gene delivery vehicle, which
by this discovery to consider employing viral genomes could carry about 10 kb of foreign DNA.

Fig. 1 Main techniques used to generate transgenic animals

Shakweer et al. Journal of Genetic Engineering and Biotechnology (2023) 21:55 Page 3 of 14

Adenoviral vectors The experiment requires a large number of embryos

Adenoviral vectors are double-stranded DNA vectors in the pronucleus stage. Thus, the average progeny
that are not enveloped. Adenoviral vectors are exten- obtained ranges from 1 to 4% when 500 to 5000 cop-
sively utilized as research tools in vitro and in small ani- ies of foreign DNA are introduced into the pronuclei.
mal models due to their relatively easy manufacture and This indicates that from a hundred injected cells, only
high levels of transgene expression [25]. Adenovirus vec- 1–4 transgenic mice are produced. In cattle, the success
tors (AdV) are extremely strong gene transfer vehicles, rate is the lowest. Because of the low rate of integration
with applications capable of holding up to 10 kb of for- of injected DNA into the genome and the restricted
eign DNA. The elimination of structural genes such as embryonic survival, producing transgenic cattle via
gag, pol, and env, which aid in the assembly of viral parti- pronuclear microinjection of DNA into fertilized
cles by the retrovirus, is a common change in this type of zygotes is difficult. The pronuclear DNA microinjection
vector [26]. technique in cows is shown in Fig. 2. Pronuclear DNA
microinjection has long been the most effective method
DNA microinjection technique for producing transgenic offspring in pigs; yet, even in
Pronuclear DNA microinjection this species, the efficacy of transgenic offspring produc-
A variety of approaches can be used to make transgenic tion is limited, with only 1% of DNA-injected embryos
animals. The most common method used to date is the resulting in transgenic animals [28]. The success rate of
microinjection of genes into the pronuclei of zygotes. the pronuclear DNA microinjection technique is low
In the 1980s, this method was first used on rabbits, and varies between species [29]. The reasons for this
pigs, and sheep and thereafter on goats and cows. divergence are unknown, although they are most likely
However, the usefulness of this approach for domes- related to changes in the DNA repair mechanism or
tic animals, is still limited [27]. The major drawback the host genome’s intrinsic DNA integration process.
of this method is that some copies of the foreign gene Furthermore, low transgenesis efficiency in domes-
are randomly integrated into the host genome, caus- tic animals may be attributable to exogenous DNA
ing transgene and host gene expression to be disrupted. purity, the method used to create the artificial molecule

Fig. 2 Showing the pronuclear DNA microinjection technique in cow

Shakweer et al. Journal of Genetic Engineering and Biotechnology (2023) 21:55 Page 4 of 14

(promoters and coding sections), and other cellular transgenic chimeric mice (Fig. 3). In these animals, the
machinery-related characteristics [29]. transgene is mosaic [31]. In the laboratory, when a leu-
kemia inhibitory factor (LIF) is given to the culture, the
Embryonic stem (ES) cells stem cells stay undifferentiated. Because LIF is absent,
The properties of stem cells are undifferentiated cells, ES cells can develop into a variety of tissues on their own
undifferentiated cells, and undifferentiated cells. 2. Have (Fig. 4).
the ability to develop into any type of cell (including
somatic and germ cells), leading to the production of a Gene transfer into gametes
full organism. Embryonic stem cells have been devel- Sperm‑mediated gene transfer technique (SMGT)
oped in vitro for a long period of time [30]. The appro- The first indication that foreign DNA might be integrated
priate DNA sequence is inserted into an in vitro culture into untreated sperm was given by Brackett et al. [32].
of embryonic stem (ES) cells using homologous recom- Lavitrano et al. [33] demonstrated for the first time that
bination. Foreign DNA can be introduced into ES cells, (a) mouse epididymal sperm can spontaneously incor-
and utilizing a selection gene, clones carrying the foreign porate plasmid DNA molecules, (b) genetically modi-
gene can be generated. These cells can be used to make fied offspring can be generated by in vitro fertilization

Fig. 3 The DNA microinjection technique using ES cells

Fig. 4 In vitro culture of embryonic stem (ES) cells

Shakweer et al. Journal of Genetic Engineering and Biotechnology (2023) 21:55 Page 5 of 14

Fig. 5 The sperm-mediated gene transfer technique

procedures using plasmid-containing sperm cells, (c) membrane was destabilized as a result, allowing foreign
exogenous DNA sequences are expressed in the progeni- DNA full access to the sperm. Also, using sperm freezing
tors, and (d) sperm-carried exogenous DNA is incorpo- and thawing, similar findings have been produced [38].
rated into the fertilized ovum (Fig. 5). Another fascinating alternative method is intracyto-
Transgenic mice, rabbits, pigs, sheep, cows, chickens, plasmic sperm injection, which involves injecting sperm
and fish have been created by incubating sperm cells with that has been treated and incubated with foreign DNA
foreign DNA and fertilizing them in vitro or in vivo [9, directly into the oocyte (ICSI). ICSI has been used to suc-
10]. Furthermore, this operation does not necessitate any cessfully transfer lengthy pieces of DNA in mice, as well
special equipment or skills, and it may be carried out in as in yeast, bacteria, and other artificial chromosomal
the field. Another fascinating feature of using sperm as constructs (YACs, BACs, and MACs) [39, 40]. Chang
DNA vectors is the concept of mass transgenesis [9, 10]. et al. [41] describe an intriguing method for produc-
In subsequent studies, the successful introduction of the ing transgenic animals that involves incubating sperm
exogenous GH expression vector into the sperm head cells with tagged foreign DNA and monoclonal antibod-
allowed for the production of GH-transgenic sheep char- ies (mAb C). mAb C is a simple protein that attaches to
acterized by a high growth rate in order to reduce the DNA via ionic interactions, allowing foreign DNA to be
meat shortage in Egypt [10]. The main binding site of connected to sperm selectively. The surface antigen on
foreign DNA in mouse sperm is mediated by a complex the sperm of all studied species, including pig, mouse,
structure of molecules from the class 2 major histocom- chicken, cow, goat, sheep, and human, is reactive to
patibility complex, which is found in the posterior area of this linker protein. It is worth noting that foreign DNA
the sperm head, according to Wu et al. [34]. In the mouse uptake mediating mechanisms are an important aspect of
seminal plasma, researchers discovered two components: the biology of sexually reproducing organisms [36].
a DNase from the seminal vesicle and a variety of foreign
DNA-binding proteins from the prostate. Exogenous
DNA sequestration has been shown to be inhibited by In vitro sperm precursors
these components [35, 36]. The production of mature sperm from stem cells is
SMGT is employed in domestic animals such as cat- known to occur at various stages of differentiation
tle and pigs by taking advantage of the farmers’ standard (Fig. 6). Sperm stem cells can be extracted, grown in vitro
artificial insemination (AI) method [37]. Fresh semen for a brief time, and then transferred into an adoptive
is taken from donor animals and cleaned several times testis. The transplanted cells continue to differentiate,
before being centrifuged to remove seminal plasma. eventually producing functioning sperm. Treatment of
Animal artificial insemination, incubation of sperm cell recipient males with busulfan, a medication that prevents
suspensions with foreign plasmid DNA (about 1 h at testis stem cell development, dramatically enhanced the
18), and dilution in suitable extender dimethyl sulfoxide amount of sperm produced by the transplanted stem
(DMSO) and Triton X-100, a mild polar detergent, were cells. This approach has been used to successfully trans-
used to improve DNA uptake in sperm [9, 10]. The sperm fer genes into stem cells while they are being cultured.
Shakweer et al. Journal of Genetic Engineering and Biotechnology (2023) 21:55 Page 6 of 14

Fig. 6 The sperm-mediated gene transfer technique (in vitro and in vivo sperm precursors)

This was accomplished with the use of a powerful retro- specific testicular cell types in vivo should provide a tool
viral vector. Transgenic mice were created at a rate of up for studying the molecular regulation of spermatogenesis
to 4% when stem cells were transplanted into busulfan- [42]. The process of gene transfer into epididymal sper-
treated recipient males. This technology could be used to matozoa by a DNA-transfectant complex injected into
investigate the biological effects of genes during the mat- the testis is being investigated. Foreign DNA inserted
uration of sperm stem cells and to create transgenic ani- into the testis, on the other hand, is thought to be quickly
mals. Extrapolation to species larger than mice is unlikely transferred to the epididymal ducts via the rete testis and
to be successful. Indeed, more highly altered cells appear efferent ducts, where it is integrated by epididymal epi-
to be required to raise the chances of colonizing testis at thelial cells and epididymal spermatozoa [43].
a significant pace. Adenovirus vector solution injected into the interstitial
space (intratesticular injection) or seminiferous tubules
Testis‑mediated gene transfer technique (TMGT) (intratubular injection) of the mouse testis is another
Other methods for creating transgenic spermatozoa have method for introducing foreign genes. The results suggest
also been explored. One of these approaches is testis- that adenovirus-mediated gene transfer may be effec-
mediated gene transfer (Fig. 7), which is a simplified form tive for transfecting testicular somatic cells, and that this
of SMGT because it does not involve IVF or embryo approach may be applicable for in vivo gene therapy for
transfer. In addition, the testis is regarded as an immune- male infertility in the future, despite the fact that sper-
privileged organ. The ability to transfer genes into matogenesis is slightly impaired and the inflammatory

Fig. 7 The sperm-mediated gene transfer technique (in vivo sperm precursors)
Shakweer et al. Journal of Genetic Engineering and Biotechnology (2023) 21:55 Page 7 of 14

response caused by these methods may present some of developmental anomalies have been documented. The
problems. The findings also suggest that TMGT could be causes of these abnormalities are unknown, however,
used for fetal gene therapy and the production of trans- they could be caused by improper or insufficient repro-
genic animals in general [44]. gramming or even issues with imprinted genes [45–47].
It may be possible to clarify the mechanisms behind
Somatic cell nuclear transfer (SCNT) these processes by having a better grasp of the systems
The nuclear transfer offspring development is an ineffi- controlling normal development. The technique involves
cient method and the successful percentage ranged from the transfer of a somatic cell nucleus to an enucleated
0.5 and 5.0%. Losses happen during pregnancy, at birth, egg’s cytoplasm where it will be reprogrammed by egg
and in the weeks and months afterward, and a number cytoplasmic components to become a zygote [48–50]. In

Fig. 8 The somatic cell nuclear transfer technique

Shakweer et al. Journal of Genetic Engineering and Biotechnology (2023) 21:55 Page 8 of 14

mammals, the zygote needs to be artificially implanted can be validated before using these cells to produce
into a surrogate mother’s uterus [51, 52]. Willadsen had genetically modified cloned animals [54]. In this proce-
his first significant success with SCNT in 1986, when dure, the DNA is randomly incorporated into the genome
he produced lambs cloned from embryo nuclei at stages by selection pressure; however, the transgenic cells may
ranging from 8 to 16 cells. This discovery piqued the be completely described (integration site, number of inte-
interest of researchers in using nuclear transfer to mul- grated copies, and transgene integrity) before being used
tiply embryos derived from high-value agricultural ani- for nuclear transfer. As a result, while “reconstructed”
mals [53]. This time-consuming procedure also opened nuclear transfer (NT) embryos have a decreased develop-
up new and exciting prospects for animal transgenesis. mental capability, the vast majority of animals born are
When the nuclei utilized in embryo reconstruction come transgenic, making this approach far more efficient than
from a cell with some genetic modification, the animals pronuclear microinjection. Transgenesis efficiency has
created by nuclear transfer could be regarded as a group increased considerably, thanks to somatic cell nuclear
of transgenic animals (addition, substitution, or altera- transfer. Only somatic cells, which are utilized to cre-
tion of some gene). In this view, transgenic embryos and ate genetically engineered animals, can perform gene
animals are defined as those produced via nuclear trans- substitution through homologous recombination at the
fer of genetically changed cells, as they carry the initial moment. Gene inactivation has been achieved in sheep
changes present in the nucleus of the donor cell from [55] and pigs [56]. The majority of animals cloned from
which the animal was derived (Fig. 8). transfected somatic cells express the transgene, according
Interest-specific exogenous genes can be transfected to results observed in cattle, sheep, goats, and pigs.
into somatic cells and then transferred to pluripotent
cells (cells of morulae or blastocysts). The progeny of the Applications of transgenic animals
chimera can inherit the exogenous gene, making them Animal production
transgenic [27]. Cultivated cells can be transfected in this Some of the practical uses of transgenesis in animal
fashion, and the insertion and expression of the transgene production include greater prolificacy and reproductive

Fig. 9 Can transgenic technology produce comparable milk volume? Small improvements in milk volume in Guzerat cows (left) using genetic
material from high-producing Holsteins (right) could have a significant impact on Brazilian beef production [68]

Table 1 An examination of transgenic livestock species and their value in animal production
Animal Genes introduced or deleted Performance criteria (consumer benefit) Reference

Bovine β and κ casein Casein protein expression has increased (improved protein content of milk) [59]
Bovine Intestinal lactase Lactose in milk is being reduced (lactose intolerant people) [60]
Bovine Lysostaphin Resistance to mastitis (reduced use of antibiotics) [61]
Bovine β-Lactoglobulin Higher milk production of this protein, as well as increased growth and illness resistance in milk- [62]
fed calves (reduced antibiotic use and improved health benefits)
Ovine Growth hormone Increased growth rates, improved feed conversion efficiency, lower carcass fatness, and higher [63]
lactation rates (leaner meat)
Ovine Myostatin In sheep, myostatin expression was reduced, and muscle mass was raised (leaner meat) [64]
Porcine Insulin-like growth factor 1 Increased growth rate and lower fat content in the carcass (leaner meat) [65]
Porcine α-Lactalbumin Piglets’ growth rate has increased, and their health has improved [66]
Shakweer et al. Journal of Genetic Engineering and Biotechnology (2023) 21:55 Page 9 of 14

performance, higher feed consumption and growth genes produced milk with 8–20% higher beta-casein
rate, improved carcass composition, improved milk levels and twice the kappa-casein levels. This work dem-
production and/or compositions, and increased dis- onstrates that by employing a transgenic approach to
ease resistance (Table 1). The most important candi- improve the functional qualities of dairy milk, a main
date gene for generating GH-transgenic farm animals component of milk in high-producing dairy cows may be
to increase the growth rate and milk production [2–4] dramatically altered.
is growth hormone. Myostatin is a negative regulator The transgenic sheep with wool keratin and keratin-
of muscle cell proliferation during fetal development. associated protein (KAP) genes could be utilized to alter
It is know that inactivating mutations in a number of the protein composition of wool fibers, resulting in fiber
species, including cattle, develop a muscle overgrowth types with improved processing and wearing qualities
phenotype [57]. Although the increased muscle can [69]. Aquaculture transgenic research is rapidly develop-
be considered a positive trait, the increase in dystocia ing on a global basis. Fish and shellfish have a high fertil-
related to the size of the calves at birth limit the utili- ity rate. Fertilization is frequently easy, and fertilized eggs
zation of these naturally occurring myostatin mutations develop outside the body, requiring no major manipu-
in production agriculture. lation, such as preimplantation. As a result, making
Genetically modifying animals to make their organs transgenic fish or shellfish is rather straightforward. In
immunologically compatible for use as human trans- Australia, the focus is on the possible use of transgenesis
plants or to improve commercial recombinant protein to control wild populations such as European carp.
output in the transgenic mammary gland was the first
animal-focused experiment [58].
Transgenic technology advancements offer the chance Environmental pollution
to modify milk’s composition or manufacture whole Phytic acid is a compound that pigs cannot produce nat-
new proteins in milk. It is possible to increase livestock urally. On the other hand, 50 to 70% of the phosphorus in
growth or survival by changing the composition of milk. grain comes from phosphorus. As a result, many farm-
To do this, transgenic animals must be developed that: (1) ers must use an enzyme called phytase to enhance pig
produce more milk; (2) produce milk with higher nutri- diets. Phytase helps pigs consume more of their nutrition
ent content; or (3) produce milk with a useful "nutriceuti- by breaking down phytic acid. Farmers pay a high pre-
cal" protein. Lactose, fat, and protein are the three main mium for the phytase enzyme, and it might be damaged
nutrients in milk. We can influence the development and or destroyed accidentally when they mix feed. To address
well-being of the growing offspring by improving any one this issue, the Enviropig was developed.
of these factors. Increased milk yield or composition can Enviropigs have salivary glands that have been geneti-
be advantageous for cattle, sheep, and goats raised for cally engineered to aid in the digestion of phosphorus in
meat production [67]. Heat-tolerant livestock breeds, feedstuffs and reduce phosphorus pollution in the envi-
like Bos Indicus cattle, are necessary for the increase of ronment [5]. The salivary glands of the transgenic pig
agricultural productivity in tropical areas. Bos Indicus synthesized phytase, removing the need for extra supple-
cow breeds do not, however, yield a lot of milk. Wean- ments or enzymes in the feed. The Enviro-Pig produces
ing weights in Brazilian cattle of the Nelore or Guzerat less phosphorus in its face by consuming more phospho-
breeds may be significantly increased by increasing milk rus [70].
production to just 2-4 litres per day (Fig. 9). Improve-
ments in weaning weights in meat-type breeds like the Medicine
Texel sheep and Boer goat can be compared in a similar Nutritional supplements and pharmaceuticals production
way. By using transgenic technology in this way, offspring Milk composition can be altered in several ways: by
may grow and survive better [68]. changing the concentration of unsaturated fatty acids,
Lactose synthesis and milk volume [67, 68] are both reducing the lactose content, removing ß-lactoglobulin,
aided by alpha-lactalbumin. The milk protein bovine or combining nutraceuticals in milk. By combining nutri-
alpha-lactalbumin was overexpressed in transgenic tional and genetic interventions, researchers are now
homozygous sows, resulting in up to 0.9 g of bovine hoping to develop “medicine milk,” rich in specific milk
alpha-lactalbumin per liter of milk produced by the sow. components that have implications for health as well as
Weight gain was increased in piglets sucking alpha-lac- treatment. In 1997, the first transgenic cow, Rosie, pro-
talbumin gilts (days 7–21 after parturition). As a result, duced human alpha-lactalbumin-enriched milk at 2.4 g
increased milk protein expression in transgenic sows may per liter. This transgenic milk is a more nutritionally bal-
aid pig lactation success. Furthermore, transgenic cows anced product than natural bovine milk and could be
with extra copies of the bovine beta- and kappa-casein given to babies or the elderly with special nutritional or
Shakweer et al. Journal of Genetic Engineering and Biotechnology (2023) 21:55 Page 10 of 14

Table 2 Some prescription drugs created by transgenic animals

Drug Disease/target Animal Reference

Alpha-lactalbumin Anti-infection Cow [80]

Human protein C Thrombosis Pig, sheep [81]
Fibrinogen Wound healing Cow &sheep [82, 83]
Glutamic acid decarboxylase Type 1 diabetes Mouse, goat [84]
Human serum albumin (HAS) Maintains blood volume Mouse, cow [85]
msp-1 Malaria Mouse [86]
CFTR Cystic fibrosis Sheep, mouse [87]
Human calcitonin Osteoporosis Rabbit [88]
Lactoferrin Tract infection, infectious arthritis Cow [89]

digestive needs [71]. This transgenic milk is a more nutri- Pharmaceutical animals
tionally balanced product than natural bovine milk and A gene encoding a pharmaceutically essential protein
could be given to babies or the elderly with special nutri- can be isolated, inserted into an expression vector, and
tional or digestive needs. then delivered into cells or organisms that produce the
protein in high quantities. Human insulin was created
Xenotransplantation from genetically modified bacteria. The vast majority of
Human life expectancy is increasing, and doctors’ abili- diabetics now receives recombinant insulin rather than
ties to transplant patients’ cells and organs are also derived pig insulin. The recombinant hormone is purer
improving. Patients who require transplants are increas- and structurally equivalent to native human insulin.
ing, while organ donors are growing far more slowly [72]. For more than a decade, the only type of human growth
Xenotransplantation is the term used to describe the hormone that has been used is one generated from bac-
transfer. The gap between the availability and demand for teria. As a result, the risk of contamination by the human
human organs is a barrier to clinical transplantation [73, prion that causes Creutzfeldt-Jakob disease has been
74]. Every day, almost 17 people pass away while awaiting eliminated [77]. This technique, while effective, quickly
an organ transplant. According to the US Government reveals its limitations. Some proteins are difficult for bac-
Information on Organ Donation and Transplantation, teria to synthesize. Others are difficult to purify because
more than 106,941 people were on the transplant wait- they become insoluble in bacteria. Furthermore, many
ing list as of October 2021, whereas only 39,000 trans- pharmaceutically essential proteins, especially human
plants were carried out in 2020 [data available at URL: proteins, are glycosylated or require posttranscriptional
https://​www.​organ​donor.​gov/​stati​stics-​stori​es/​stati​stics.​ alteration in order to function physiologically. Bacteria
html (accessed September 2021)]. Xenotransplantation and recombinant yeast are unable to progress through
might be a good solution to this major issue. Although the majority of these stages.
the surgical part of the procedure was frequently suc- Genetically engineered animal cells are being
cessful, the xenogeneic foreign organs were always vio- exploited as a source of recombinant proteins. It
lently rejected and killed. As a result, the technology became possible to create transgenic farm animals
aims to create humanized organs from pigs by prevent- larger than mice (Table 2) (rabbits, pigs, and sheep)
ing organ rejection brought on by physiological differ- that secrete foreign proteins in their blood, milk, and
ences between humans and pigs as well as the spread of other bodily fluids. Milk protein gene promoters were
illnesses with genetic origins [75, 76]. A pig protein that fused to the coding area of the sheep β-lactoglobulin
can lead to donor rejection currently limits the utilization gene, as well as human tissue plasminogen activator
of xenotransplantation, although research is being done to the coding β-lactoglobulin gene [78]. In an experi-
to substitute the pig protein with a human protein [67]. mental setting, this method allowed the secretion of
Concerns with welfare, ethics, and clinical and safety 100 foreign proteins in milk; milk with higher casein
considerations are additional difficulties. Future solutions levels, which is good for making cheese, or milk with
to the issue of transgenic organs may involve improving particular qualities to fill population gaps, such as
the supply of National Health Services and promoting lactose-free milk for the Asian market, milk without
tissue donation and stem cell regeneration [76]. β-lactoglobulin for consumers with allergies, or milk
containing the human β-lactoferrin protein to ensure
Shakweer et al. Journal of Genetic Engineering and Biotechnology (2023) 21:55 Page 11 of 14

the health of newborns [79]. A number of them are cost of the transgenic process, we can only obtain a small
detected in large amounts in rabbit, sheep, goat, and number of animals. Because of this, backcrossing is a dif-
cow milk and are clinically tested. Human a-glucosi- ficult process that must be used to reintroduce such ani-
dase, generated from rabbit milk, was one of the pro- mals into the herd. To make this process economically
teins that improved the clinical state of babies with feasible, it is essential to possess in-depth knowledge of
Pompe disease. Collagen, fibrinogen, spider silk, and the most recent advancements in assisted reproduction
EC superoxide dismutase are just a few of the complex techniques, such as artificial insemination combined with
foreign proteins that may be produced by the mam- embryo transfer or in vitro embryo formation [79, 92].
mary gland [78]. The only real issue with using transgenic animals to study
human diseases is ethical. The same is true for animals
used as sources of proteins or organs for pharmaceu-
Industry ticals because the manipulation of embryos can have a
Nexia Biotechnologies Inc. has developed a strain of negative impact on animal welfare [93, 94]. Commissions
with extensive experience in this area with conventional
lactate early (BELE®), decreasing transgenic protein
dwarf goats from West Africa that naturally breed and
chemical medications are evaluating the medical issues
caused by the usage of pharmaceutical proteins [72, 95].
tional goats. Male BELE® goats, for example, reach sex-
production time compared to sheep, cows, and conven-
There is probably no damage to the environment from
ual maturity at the age of 15 weeks, whereas traditional transgenic animals. More dangers may be posed by trans-
male goats reach sexual maturity at the age of 30 weeks. genic fish and live virus-based vaccinations, which raise
This reduces the time it takes to produce a transgenic complex issues for environmental risk assessment [96].
herd. Clinical trials and product commercialization According to Muir, and Howard, [97], growth hormone
can start sooner due to the shorter time between lab (GH)-transgenic fish are rapidly growing and more sexu-
amounts and production quantities of protein [52]. ally mature but are more fragile and have shorter lifes-
Two Canadian scientists spliced spider genes into the pans than the controls. The quickly expanding transgenic
cells of goats in 2001. The goats began to make silk with fish that was released into the ocean afterward could be
their milk, which is the strongest material in the world to blame for the local extinction of the species. Although
and named “Bio-Steel.” Scientists can manufacture a unlikely, this prospect cannot be discounted, and the reg-
light, durable, and flexible material by separating poly- ulatory bodies have not, up until this point, approved the
mer strands from milk and weaving them into thread, breeding of fast-growing fish using the current methods.
which might be utilized in military uniforms, medical The issue could be resolved by sterilization of females
micro sutures, and tennis racket strings, among other and breeding of females only or completely isolating fish
applications [48]. farms. Biosafety organizations, on the other hand, may
allow humans to consume rapidly growing fish but not
The risks of the application of transgenic animals reproduce them. Assessing the impact of transgenic ani-
The main environmental issue associated with transgenic mals on biodiversity requires knowledge. So that the state
animals is the possibility of their escape. The risks differ can evaluate the impacts of potential transgenic animals
significantly depending on the transgene and the spe- on biodiversity, this information must be updated on a
cies. Some farm animals cannot live in the wild because regular basis [97].
they are confined. Transgenic animals are mostly used to
study genes and biological functions. Transgenic animals Conclusion
might also be a useful model for investigating human and Genetic engineering is used to incorporate foreign genes
animal diseases and testing experimental medication or into the animal genome so that they can be inherited
a source of human organs [90]. The technology involved and expressed by offspring via transgenic animal tech-
in the production of transgenic animals holds great nologies. To address the current and future demands
promise for both agriculture and biomedicine but also of the human race, transgenic animals are required in
has potential risks. So, it is important for scientists to agricultural techniques, food supply development, and
become engaged in the discussion and consideration of food consumption management. Moreover, with the
ethical issues and concerns surrounding the implemen- development of disease-resistant animals and other
tation of this work and the effects on the environment, approaches for enhancing animal production capacity,
farmers, and consumers; the use of this technology is animal transgenesis has the potential to replace tradi-
not simple, efficient, or inexpensive [91]. The "biosafety" tional drug use in the future. It was also used to improve
field of research to manage the ecological effects of trans- human health by filling organ shortages and producing
genic animals has recently emerged [90]. Due to the high vital pharmaceuticals to treat human illnesses. Animal
Shakweer et al. Journal of Genetic Engineering and Biotechnology (2023) 21:55 Page 12 of 14

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Abbreviations
kopoulos R (eds) Geo ENV VI-geostatistics for environmental applications.
CP Crude protein
Quantitative geology and geostatistics, Vol. 15. Springer, Dordrecht, pp
CF Crude fiber
59–70
EE Ether extract
7. Margawati ET (2003) Transgenic animals: their benefits to human welfare.
TDN Total digestible nutrient
Action Bioscience, USA
DCP Digestible crude protein
8. Gordon I (1996) Controlled reproduction in cattle and buffalo: production
DMI Dry matter intake
of transgenic animal Vol. 1. Cab International, Wallingford, pp 338–372
LBW Live body weight
9. Shakweer WME, Hafez YM, El-Sayed AA, Awadalla IM, Mohamed MI (2017)
TBWG Total body weight gain
Construction of ovine GH-pmKate2N expression vector and its uptake
ADG Average daily gain
by ovine spermatozoa using different methods. J Genet Eng Biotechnol
TDNI Total digestible nutrient intake
15:13–21
DCPI Digestible crude protein intake
10. Shakweer WME, Hafez YM, El-Sayed A, Dessouki ShM, Awadalla IM,
AIA Acid insoluble ash
Mohamed MI (2019) Uptake of exogenous bovine GH–pmKate2– N
OM Organic matter
expression vector by rams spermatozoa. Bull Nati Rese Cent 43:96.
NFE Nitrogen-free extract
https://​doi.​org/​10.​1186/​s42269-​019-​0136-4
NDF Neutral detergent fiber
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ADF Acid detergent fiber
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ADL Acid detergent lignin
theri​ogeno​logy.​2005.​06.​009
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