Dear Editor,
Golden Syrian hamsters are small rodents, but they display many features that resemble
the physiology and metabolism of humans. Hamsters have been widely used in many research
areas, including carcinogenesis
1
, reproduction
2
, virology
3
, diabetes
4
and cardiovascular diseases
5
. With respect to lipid and glucose metabolism, hamsters, like humans, exhibit high
levels of cholesteryl ester transport protein (CETP), intestinal-only ApoB editing,
low levels of hepatic low-density lipoprotein (LDL) receptor activity
6
and a high glycemic response to dietary fructose
7
, all of which are not observed in other rodents such as mice and rats. Consequently,
hamsters, like humans, exhibit enhanced susceptibility to atherosclerosis (AS) and
diabetes
8
, which led to the widespread use of hamsters in studies on AS and diabetes.
In the past 2-3 decades, due to the fast development of transgenic and knockout mice,
hamsters were gradually replaced by these mouse models. However, due to multiple differences
between mice and humans with respect to physiology and metabolism, the use of gene-manipulated
mice has limited value in disease modeling and pathophysiological studies. Extensive
literature search has revealed an absence of reports on genetically manipulated hamster
models. To capitalize on the special metabolic features of hamsters, we aim to generate
gene-manipulated hamsters as an alternate rodent model for general applications. As
the initial step to create a genetically manipulated hamster, we utilized a highly
efficient lentiviral vector to generate transgenic hamsters expressing enhanced green
fluorescent protein (eGFP).
By modifying and optimizing the protocols for producing transgenic mice and rabbits
in our laboratory
9,10
, we developed a specific procedure for hamster superovulation, fertilized egg harvesting,
perivitelline space microinjection and embryo transfer. After the successful culture
of fertilized hamster eggs that developed into 4- and 8-cell embryos in vitro (Figure
1A), we implanted these embryos into pseudopregnant females. We obtained 7-10 pups/litter
in 4 out of 7 surrogate mothers. Next, we microinjected 50-100 picoliters of a lentiviral
eGFP vector (Figure 1B) at a titer of 2 × 109 titer units/ml into the perivitelline
space of the fertilized eggs to generate transgenic hamsters that express eGFP.
A total of 6 out of 32 live-born pups from 5 surrogate mothers each receiving 30-40
microinjected eggs were identified as being eGFP-positive by PCR genotyping, and 5
of the pups were further validated by Southern blot analysis (Figure 1C and 1D). Of
the 5 positive pups validated by Southern blotting, 2 of them (males) expressed eGFP
in the exposed skin area as determined by direct fluorescence imaging (Figure 1E).
Transgenic lines were then established by breeding the 2 founders (F0) with non-transgenic
females. Among the progenies from 3 litters, 21% of the animals were eGFP-positive
as determined by direct fluorescence imaging. Representative fluorescence images of
the first generation (F1) pups from one of the 3 litters are shown in Figure 1F. All
the examined organs from one eGFP-positive F1 pup showed strong-to-moderate levels
of green fluorescence, which include the liver, kidney, heart, skeletal muscle, lung,
brain, white/brown adipose tissues, adrenal glands and eyes; however, no fluorescence
was observed in these organs of the littermate control (Figure 1G and data not shown).
The peritoneal macrophages and bone marrow cells from another eGFP-transgenic F1 hamster
also demonstrated an approximately 70% eGFP-positive cell population as analyzed by
FACS (Figure 1H).
With the establishment of transgenic hamster model in the present study, it is now
possible to generate small rodent disease models that recapitulate human pathogenesis.
For example, generation of a transgenic hamster overexpressing proprotein convertase
subtilisin/kexin type 9 (PCSK9), a protein involved in cholesterol homeostasis by
inducing LDL receptor degradation, will be highly desirable. Although mice and pigs
overexpressing PCSK9 developed hypercholesterolemia and AS
11
, these animals, unlike humans, do not express CETP, a critical factor involved in
lipid transport and AS development.
Along with the recent development of genome-editing methods such as TALENs (transcription
activator-like effector nucleases) and the CRISPR/Cas system (clustered regularly
interspaced short palindromic repeats/CRISPR-associated system)
12
, the generation of gene-targeted hamster models is thus warranted in conjunction
with the hamster embryo manipulation method optimized in the present study. These
hamster models will be utilized extensively in studies for metabolic cardiovascular
diseases and will become the candidate models when genomics and proteomics tools are
fully developed for this species in the future. Genetically altered hamsters may potentially
replace mice as the mainstream animal model for metabolic cardiovascular research.
Therefore, the present study will promote the use of genetically engineered hamsters
as disease models. These models might recapitulate many features of human metabolic
disorders while simultaneously retaining the ease of handling, the simplicity of management
and the cost efficiency of small rodents as compared to genetically engineered large
animals such as mini-pigs
13
or non-human primates
14
.
Detailed methods are described in the Supplementary information, Data S1.