Transgenic Mice Models

Applied StemCell has a strong service portfoilio for generating transgenic mouse models using its site-specific CRISPR and TARGATT™ technologies, including conditional knockout and large fragment knock-in (up to 20 kb) models. We are also very experienced in generating mouse models using traditional homologous recombination via embryonic stem cells (ESCs).

Our animal specialists can also generate transgenic mouse models using different approaches to meet your specific needs.

(1) Bacterial artificial chromosome (BAC) to insert the complete foreign gene along with the promoter, introns and other required regulatory elements for gene expression

(2) Random microinjection of the foreign gene into the pronucleus of a fertilized mouse egg

Please note that the expression of the transgene and the subsequent transgenic mouse phenotype may vary from line to line due to the nature of random gene insertion. We recommend generating multiple independent transgenic mouse lines in order to interpret the experimental results.

 

Generation of transgenic mice by BAC cloning

Step 1: Design and cloning of gene of interest, promoter, and regulatory elements into BAC vector

Step 2: Pronuclear microinjection of vector DNA into mouse embryos

Step 3: Transfer of injected embryo into recipient mothers

Step 4: Genotyping of offspring to identify potential transgenic founders

Step 5: Breeding to F1 or F2 progenies (optional)

 

Generation of transgenic mice by random insertion

Step 1: Cloning of cDNA, promoter of interest, and other elements

Step 2: Construction of the transgene vector

Step 3: Pronuclear microinjection of vector DNA into mouse embryos

Step 4: Transfer of injected embryo into recipient mothers

Step 5: Genotyping of offspring to identify potential transgenic founders

Step 6: Breeding to F1 or F2 progenies (optional)

 

Case Studies

Case Studies for Transgenic Mice Generated by Pronuclear Microinjection

Case #1: Knock-in of a cell proliferation inhibitor gene in C57BL6/N mice by microinjection of DNA of interest directly into the pronucleus of a fertilized mouse egg.

Casestudy-Random Tg mice-case#1

Figure: PCR confirmation of gene knock-in by random insertion into C57BL6/N mice. Twelve pups were born from two rounds of microinjection. Two pups (lane #6, lane #11) were confirmed to carry the inhibitor gene by PCR; lanes #2-13: twelve F0 pups; lane #1: GeneRulerTM 1kb plus DNA ladder; lane #14: B6/N negative control.

Case #2: Knock-in of a NAD/NADH related enzyme gene in C57BL6/N mice by microinjection of DNA of interest directly into the pronucleus of a fertilized mouse egg.

casestudy-random Tg mice-case2

Figure: PCR confirmation of gene knock-in by random insertion in C57BLB6/N mice. Ten pups were born from one microinjection experiment. Seven of them (Lanes #1, 2, 6, 7, 8, 9 and 10) were confirmed to have the enzyme gene knock-in by PCR; lanes #1-10: ten F0 pups; lane #11: GeneRulerTM 1kb plus DNA Ladder; lane #12: B6/N negative control.


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