Knockout Mouse Catalog | Cyagen APAC

Due to the significant genetic similarity between humans and mice, transgenic mice are important tools for scientists researching heritable traits and diseases in human populations. In traditional transgenics, the human gene (packaged in a transgenic construct) can be added to the mouse genome with a simple pronuclear injection into the male pronucleus. Transgenic model organisms are helpful in research for studying human diseases and genetic disorders, such as heart disease, obesity, Parkinson’s disease, and substance abuse disorders – exemplified by countless research publications and therapeutic progress enabled by transgenic models.

Watch our newest video to learn about the steps involved in pronuclear injection (PNI)-mediated generation of transgenic mouse models, which are used by scientists to study a wide range of human diseases.


Video: The Basics of Transgenic Mice – Pronuclear Injection (PNI)


Transgenic Research and Transgenic Pets

Scientists have already created many amazing transgenic animals, some of which are even commercially available. GloFish - introduced to the US market in 2003 - are one of the earliest examples of transgenic pets. These zebrafish have been genetically engineered to contain genes from other organisms, such as jellyfish or sea anemones, that result in expression of fluorescent proteins. Additional species of fish have been developed to express a range of colors, using genes extracted from a variety of organisms which naturally produce fluorescent proteins. These fluorescent transgenic pets have a strong basis in development of model organisms for research - the ability to express fluorescent proteins under certain conditions has been utilized in many genetic engineering studies.

What are Transgenic Organisms?

A Transgenic Organism is an individual of a species that contains a gene from another individual of the same species or from another species. These genes from another species are called transgenes. Transgenesis is the process used by scientists to introduce a gene from one animal to another: This process has three main steps:

  1. In the first step, Identification, the gene for a specific trait is identified. 
  2. In the isolation step, the vectors are created by breaking the cells, collecting the target DNA sequence, replicating it, and inserting it into the bacterial genome. 
  3. In the final step, Transformation, the vector containing the transgene is inserted into the target animal. For some organisms like mice, the most efficient way to transfer DNA into the cells is microinjection into the male pronucleus. This is called pronuclear injection (PNI) and it is the most common method of producing transgenic mice today.

Biology Basics: What are Oocytes?

Before we go over the steps involved in pronuclear injections (PNIs), let us first cover the biological basics of the process. As you may know, all female mammals are born with all the reproductive cells inside of them that they will produce for their entire lives - these are called oocytes. The fertilization of oocytes offer scientists many opportunities to take advantage of important cellular mechanisms. Within 24 hours of fertilization the fertilized oocytes will have developed into zygotes, at which point two pronuclei appear inside of the embryo.

Steps of Pronuclear Injection (PNI)-based Transgenic Mouse Model Generation

Let us move on to the steps of a pronuclear injection (PNI) and what happens inside the mouse genome as these steps progress:

  1. Two wild type (WT) mice - which have phenotypes/genetics reflecting the ‘normal’ background strain - are mated together. 
  2. The fertilized oocytes are removed from the female reproductive tract. 
  3. Since bacteria carry circular genomes, restriction enzymes are used to splice apart the DNA in from bacterial vector to linearize it. 
  4. Pronuclear injection (PNI) of the linear transgene is administered into the male pronucleus of the wild type (WT) zygote. Upon inoculation of the WT zygote, several copies of the injected DNA will integrate into a random location on the chromosome. 
  5. In the next step, Pseudopregnancy is induced in Female mice using a progesterone injection.
  6. The zygotes containing the foreign DNA (transgene construct) are now injected into this mouse which gives birth to mice which may or may not contain the transgene.


To determine which offspring contain the gene, snippets of their tails are tested using PCR genotyping. The mice that test positive for the transgene can be mated with other mice to establish lines of transgenic mice. 


Random Integration of Transgene Constructs

To obtain transgenic mice via pronuclear injection (PNI), scientists must inject many zygotes with the transgene before implanting them into a surrogate mother. Although many of them will die, each one of them possesses the potential to turn into its own transgenic mouse. In a Traditional pronuclear injection (PNI), each of these zygotes will undergo a totally random integration of the transgene as it finds its way into the animal's genome. As noted in step 4 (above) of the PNI-based Transgenic mouse model generation process, injection of the male pronucleus results in random integration of the transgene construct. Scientists think this may occur as DNA repair enzymes seek out and repair broken ends of DNA. However, the exact mechanisms for random integration have not yet been determined. This randomness includes the location of integration, how many different sites undergo integration, and how many copies of the gene are integrated into each site. These three major factors will lead to great varieties in the expression level of the offspring.


Transgenic Mouse Model Services from Cyagen

As you can see, using standard pronuclear injection (PNI) is a simple way to get transgenic mice, but it leads to big variations in levels of gene expression in the offspring. For this reason, scientists have developed other more efficient methods of creating transgenic mice, such as PiggyBac transgenesis, which we will talk about in our next video.

In addition, the rapidly growing capabilities of CRISPR- and embryonic stem (ES) cell-mediated technologies, such as our proprietary TurboKnockout® Gene Targeting services, have enabled the production of transgenic, large fragment knock-in (LFKI), and humanized mouse models.

Cyagen’s Transgenic Rodent Model Generation Capabilities

Cyagen provides comprehensive model generation services for all your transgenic mouse, mouse embryo, and rat model needs, including:

  • Plasmid-based transgenics
  • BAC-based transgenics
  • PiggyBac-based transgenics
  • PiggyBac-on-BAC transgenics
  • Rosa26 (Safe Harbor) targeted transgenics

The table below offers a brief overview of the general capabilities provided by our leading service options for generating transgenic rodent models for research.


Comparison of Transgenic Model Generation Techniques and Capabilities


Regular Transgenic

PiggyBac Transgenic

Rosa26 Targeted Transgenic


Random, multicopy integration

Random, single copy per integration site

Single copy transgene targeted to Rosa26 safe harbor locus

Vector construction

Transgenic plasmid or BAC

Transgenic plasmid or BAC

Targeting vector + gRNAs

Expression pattern

Variable expression in founders

More consistent expression in founders


Most consistent expression


Endogenous effects

Can disrupt endogenous gene expression

Less likely to disrupt endogenous gene expression

Safe harbor site (SHS) does not disrupt endogenous gene expression




Options: Heterozygous and Homozygous


2-5 months

2-5 months

6-9 months (F1)


Mouse, mouse embryos, rats

Donor background

Mouse strains: C57BL/6, FVB

Rat strains: Sprague-Dawley (SD), Long Evans

Note: Other strains available upon request.


Advantages of PiggyBac Transgenic Services


Our proprietary PiggyBac transgenic method has the following advantages over other transgenic approaches:


Single-copy integration: Avoids potential gene silencing from multiple copies per integration site

Defined region of integration: No loss of transgene sequence (TTAA, transcription unit)

Reliability: More consistent expression pattern compared to plasmid-based transgenics

Economical: Cost and turnaround time comparable to plasmid-based transgenics


Large Fragment Knock-in (LFKI) Mouse Model Capabilities

Cyagen has successfully generated large fragment knock-in mouse models using TurboKnockout® or CRISPR, across both endogenous gene loci and Rosa26 loci. We provide a complete range of services, from generation of the engineered parental ES cell line through delivery of research-ready custom mouse models.

Using data from thousands of knock-in mouse model projects completed by our team, we have collected new information demonstrating how our gene editing technologies push the boundaries of modifying large genomic regions.  Below, we have outlined Cyagen’s large fragment knock-in (LFKI) capabilities across our gene editing technologies.

Comparison of TurboKnockout® and CRISPR Methods for LFKI Model Generation



CRISPR/Cas9 Gene Editing


Homologous recombination in ESC by our proprietary TurboKnockout® technology

CRISPR/Cas9 nuclease mediated gene targeting by pronuclear injection


Conditional knockout
Point mutation
Large fragment knockin

Conditional knockout
Point Mutation
Large fragment knockin
Global knockout

Knock-in (KI) fragment size limits

~20 kb per round of gene targeting
RMCE Humanization: ~ 300 kb

Endogenous: ~15 kb
Rosa26/H11: ~12 kb

Conditional Knockout (cKO)

Single target: ~7 kb
Double target: ~100 kb

Donor vector: ~7 kb
ssDNA: ~100 kb

Donor backgrounds

Mouse strains: C57BL/6, BALB/c

Mouse strains: C57BL/6, FVB
Rat strains: Sprague-Dawley (SD), Long Evans

Turnaround time

6-8 months

5-7 months


Contact Cyagen for Transgenic Model Generation Support

From strategy design through to delivery of research-ready custom mouse models, Cyagen offers complete outsourcing for all your animal model needs. Cyagen’s gene editing services are unparalleled in efficiency of developing rodent models with a guaranteed genotype. We even offer price matching to help ensure researchers get the best deal for their study.

Contact us to perform your entire transgenic project - from initial strategy design and DNA vector construction, all the way through breeding – we deliver research-ready transgenic rodent models for guaranteed results.



  1. Ford, Emma, et al. The First Mitotic Division of the Human Embryo Is Highly Error-Prone, Prelights, 28 July 2020, 
  2. “Chapter 18: Manipulating the Genomes of Eukaryotes.” Genetics: from Genes to Genomes, by Leland Hartwell et al., McGraw-Hill Education, 2018. 
  3. “Embryo Culture.” Sigma, 
  4. “TRANSGENESIS.” Pathwayz,



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