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NHEJ Non-homologous end joining
sgRNA Single-guide RNA
Manipulating the genome of laboratory animals, particularly rodents, has been of
instrumental importance for innumerable biomedical advancements. In vivo gene
function, cell differentiation, development, disease progression, and drug discovery
are just a few of the scientific mysteries that genetically modified animals have
helped to elucidate. Nearly 40 years since the development of the first transgenic
mouse [ 1 ], transgenic and gene targeting methods have been refined, and many new
techniques have been introduced. A transgenic core facility has been the major place
to perform these techniques to provide the animal model production service for
researchers. The latest genome-editing technology, clustered regularly interspaced
short palindromic repeats (CRISPR)/Cas9 system, offers a previously unattainable
speed and efficiency in targeted gene mutagenesis that has revolutionized the prac-
tice of rodent model generation and is being implemented in transgenic facilities
around the world.
Genetically engineered laboratory animals can be broadly divided into threecategories: (1) transgenic animals that carry a foreign DNA fragment, known as a
transgene, introduced into the genome in a randomly integrated or targeted (e.g.
recombinase-mediated cassette exchange) fashion; (2) targeted mutant animals
where the mutation (e.g. gene disruption, replacement, or insertion) is introduced
to a specific locus via an embryonic stem (ES) cells approach, followed by chime-
ric animal production, or a programmable nuclease-mediated gene editing tech-
nique; (3) animals carry random point mutations induced by chemicals (e.g.
ethylnitrosourea). Transgenic and targeted mutant animals, generated by trans-
genic core facilities via specialized zygote microinjection techniques, are the major
animal models used in research. The most common zygote microinjection tech-
niques include pronuclear injection, ES cell injection, and cytoplasmic injection.
While these injection methods have not changed much since their inception, they
have been retrofitted for new uses, particularly nuclease-mediated genome
engineering.
4.1 Pronuclear Microinjection
The pronucleus is the nucleus of a spermatozoon or an ovum containing the hap-
loid paternal or maternal DNA. After fertilization, at which a sperm enters an egg,
two pronuclei are present in the resulting one-cell embryo and eventually fuse to
form a diploid nucleus. Foreign DNA or the genome editing materials are back-
loaded into a filamented glass capillary needle with a fine tip (~0.5 μm in diameter)
C.L. Yuan and Y.-C. Hu