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Conditional Knockout Mouse Models

Conditional Knockout Mouse Models

Fast, Predictable, and Guaranteed.

Control where and when your gene of interest is knocked out with a conditional knockout mouse model from ingenious. When you receive your new conditional line, it can be used in conjunction with hundreds of existing Cre lines for precise regulation. Generating one mouse line with the potential for producing multiple derivative mouse models is a great way to save time and money.

Our Four Conditional Knockout Model Variations:

➡️ Standard Conditional KO➡️ Conditional KO w/ Reporter
➡️ KO First with Conditional Potential➡️ TruView Conditional Knockout™

Standard Conditional Knockout

 A tissue-specific knockout mouse defines an animal model in which a gene of interest is “floxed” and thus inactivatable in specific cell types in a certain tissue. Other cell types and tissues exhibit an unmodified, functional gene expression. This gene inactivation is achieved by the Cre loxP system, where the floxed mouse line is mated with a tissue- or cell type-specific Cre-deleter mouse line. Such models also give access to whole body knockouts.

Conditional KO w/ Reporter

Adding a reporter gene to your conditional knockout makes your research more certain by enabling you to visualize where your gene is inactivated. This gives you confirmation at the level of single cells that recombination has knocked out the target gene. Different strategies for incorporating reporters are available, depending on the genomic structure of your gene of interest.

Knockout-First Mouse Model

Knockout-first mice – with or without a reporter – can be used in gene rescue experiments and can also include conditional knockout potential. The KO-first allele overcomes the limitations of constitutively-expressed mutations, flexibly producing reporter knockouts, conditional knockouts, and null alleles through exposure to site-specific cyclization recombination (Cre) and flippase (FLP) recombinases – such as in crosses to FLP and Cre mice.

TruView Conditional Knockout™

To address the critical need of researchers to verify the loxP-mediated recombination of their conditional knockout models, and to do so in a reliable manner without increasing timelines, ingenious has developed the TruView Conditional Knockout™ system.

Conditional+Reversible Design

It is now possible to switch gene expression multiple times, in a single mouse model. Our conditional+reversible design gives you the control you need for more complex experiments.

The figure above illustrates that this design allows for initial expression of cDNA #1 in place of the wild-type (WT) sequence, then conditional expression of cDNA #2 in cells where Cre is active, and finally reversion to expression of the gene’s wild-type sequence.

How Conditional Knockout Mice Work

With conditional knockout mice from ingenious, you can control where knockout occurs by crossing it with a recombinase-expressing line, available in repositories. Cre recombinase is the enzyme most commonly used for conditional genetic rearrangements. It recognizes specific sequences known as lox sequences, e.g. loxP sites. Other recombinase/recognition sequence pairs are also used such as FLP/FRT and Dre/rox.

A conditional approach allows for tissue specific deletion in mice, avoiding potential embryonic lethality from constitutive knockout. Using Cre lines with drug-inducible activity expands your options to include temporal control of your knockout. There are many Cre mice to choose from such as tissue specific, inducible, and ingenious’ own Split-Cre for generating Cre mouse models that are not currently available. The placement of loxP sites can be tricky when considering promoter and regulatory regions, ingenious experts can help alleviate this concern. Contact us today and our experienced scientists will analyze your gene of interest and develop a targeting strategy that is perfect for your research.

The Future of Conditional Knockout Mouse Models

Our technologies can enhance the utility of your conditional knockout (cKO) line.

ingenious’ proprietary TruView Conditional Knockout™ technology can activate expression of a reporter gene in cells where your gene is knocked out independent of native gene promoter activity.

Enhance your conditional knockout mice by adding a reporter to label affected cells, and for visualizing where your gene has been knocked out on the cellular level.

Selected Citations: conditional mouse models from ingenious

Lee J, Chae S, Nan J, Do Koo Y, Lee S, Park Y, Hwang D, Han W, Lee D, Kim Y, Chung S, Park K. 2022. SENP2 suppresses browning of white adipose tissues by de-conjugating SUMO from C/EBPβ. Cell Reports, 38(8): e3.

Rosier K, McDevitt MT, Smet J, Floyd BJ, Verschoore M, Marcaida MJ, Bingman CA, Lemmens I, Dal Peraro M, Tavernier J, Cravatt BF, Gounko NV, Vints K, Monnens Y, Bhalla K, Aerts L, Rashan EH, Vanlander AV, Van Coster R, Régal L, Pagliarini DJ, Creemers JWM. 2021. Prolyl endopeptidase-like is a (thio)esterase involved in mitochondrial respiratory chain function. Science 24(12): 103460.

Vacher CM, Lacaille H, O’Reilly JJ, Salzbank J, Bakalar D, Sebaoui S, Liere P, Clarkson-Paredes C, Sasaki T, Sathyanesan A, Kratimenos P, Ellegood J, Lerch JP, Imamura Y, Popratiloff A, Hashimoto-Torii K, Gallo V, Schumacher M, Penn AA. 2021. Placental endocrine function shapes cerebellar development and social behavior. Nat Neurosci 24(10): 1392-1401.

TAM receptors regulate multiple features of microglial physiology

Published in the journal Nature, a client examined the effects of the TAM receptor tyrosine kinases Mer and Axl on regulating microglia in the central nervous system, using conditional knockout mice generated by ingenious. Through the use of these knockout mice, our client was able to determine that TAM receptors play a significant role in how microglia regularly clears dead brain cells and responds to brain damage. This study has identified TAM receptors as possible targets to prevent the development of central nervous system (CNS) disease, as well as Parkinson’s disease.

Fourgeaud L, Traves PG, Tufail Y, Leal-Bailey H, Lew ED, Burrola PG, Callaway P, Zagorska A, Rothlin CV, Nimmerjahn A, Lemke G. 2016. TAM receptors regulate multiple features of microglial physiologyNature 532: 240-244.

PIK3IP1/TrIP restricts activation of T cells through inhibition of PI3K/Akt

Phosphatidylinositide-3-kinases (PI3Ks) play important intracellular signaling roles in immune cells, with over-activation of PI3K leading to cancer and autoimmune disease. PI3K-interacting protein-1 (PIK3IP1/TrIP) is a T-cell transmembrane protein proposed to regulate PI3K. Using a conditional TrIP mouse model created by ingenious, the lab of Lawrence P. Kane at the University of Pittsburgh showed that TrIP knockout enhances T cell activation via the PI3K pathway, uncovering TrIP as an inhibitor of PI3K and placing TrIP as a novel target for immune modulation.

Uche UU, Piccirillo AR, Kataoka S, Grebinoski SJ, D’Cruz LM, Kane LP. 2018. PIK3IP1/TrIP restricts activation of T cells through inhibition of PI3K/AktJ Exp Med215(12): 3165-3179.

Deletion of DDB1- and CUL4- associated factor-17 (Dcaf17) gene causes spermatogenesis defects and male infertility in mice

A conditional knockout mouse model by ingenious was used to investigate the function, role, and necessity of DCAF17 in cellular processes. More specifically, our client studied how the deletion of Dcaf17 in different tissues can affect several processes in vivo. They found that while female fertility was unaffected by this disruption, it impacted normal sperm development, resulting in male infertility. This suggests that DCAF17 plays a crucial role in spermiogenesis.

Ali A, Mistry BV, Ahmed HA, Abdulla R, Amer HA, Prince A, Alazami AM, Alkuraya FS, Assiri A. 2018. Deletion of DDB1- and CUL4- associated factor-17 (Dcaf17) gene causes spermatogenesis defects and male infertility in miceSci Rep 8(1): 9202.

MICU1 regulation of mitochondrial Ca(2+) uptake dictates survival and tissue regeneration

To study the previously unknown role of MICU1 (Mitochondrial Calcium Uptake 1), a client used a conditional knockout mouse generated by ingenious. It had been previously established that MICU1 controls mitochondrial Ca(2+) uptake. Because knocking out MICU1 in all tissues can be lethal, ingenious created cKO mice by using the Cre-loxP system to target and knock out exon 3 of the MICU1 gene. With this mouse, our client was able to conclude that MICU1 is essential for tissue repair, as its absence can cause necrosis and prevent liver regeneration. It was also discovered in this research study that a whole body knockout of MICU1 results in lethality in newly born pups.

Antony AN, Paillard M, Moffat C, Juskeviciute E, Correnti J, Bolon B, Rubin E, Csordas G, Seifert EL, Hoek JB, Hajnóczky G. 2016. MICU1 regulation of mitochondrial Ca(2+) uptake dictates survival and tissue regenerationNat Commun 7: 10955.

Altered Neocortical Gene Expression, Brain Overgrowth and Functional Over-Connectivity in Chd8 Haploinsufficient Mice

Because CHD8 mutations play a role in the development of autism spectrum disorder (ASD) as a high risk factor, a client generated a conditional knockout mouse model with ingenious to examine the Chd8 gene. To target the gene, ingenious scientists floxed exon 3. After linearization, the construct was electroporated into C57BL/6 ES cells. The resulting clones were injected into Balb/c blastocysts to produce chimeras, which were then bred with FLpe deleter mice to create Chd8flox/+ mice. These mice were then crossed with β-actinCre mice to produce β-actinCre;Chd8+/−. In order to remove the Cre transgene and generate a Chd8+/− conditional knockout line, they were bred with C57BL/6J mice.

By using this cKO mouse, our client was able to observe that Chd8+/− mice experienced anomalies in brain development and long-range brain wiring, resulting in brain over-connectivity. This over-connectivity can be linked to symptoms of ASD, suggesting that Chd8 plays a role in a form of the disorder.

Suetterlin P, Hurley S, Mohan C, Riegman KLH, Pagani M, Caruso A, Ellegood J, Galbusera A, Crespo-Enriquez I, Michetti C, Yee Y, Ellingford R, Brock O, Delogu A, Francis-West P, Lerch JP, Scattoni ML, Gozzi A, Fernandes C, Basson MA. 2018. Altered Neocortical Gene Expression, Brain Overgrowth and Functional Over-Connectivity in Chd8 Haploinsufficient MiceCereb Cortex 28(6): 2192-2206.

Frequently Asked Questions:

What is the difference between knockout and conditional knockout mice?

With a traditional or conventional knockout, a target gene is permanently inactivated in all tissues at all time points, while a conditional knockout allows for the target gene to be turned off at specific times or in specific tissues.

RELATED: Conventional vs. Conditional Knockout Mice: The Essentials

How do you make a conditional knockout mouse?

Knockout mice are generated by flanking sequences of importance (e.g., an exon) with LoxP sites and introducing an FRT flanked reporter cassette into an intron of the target gene.

What is Cre-Lox knockout system in mice?

The Cre-loxP system is widely used as a tool for generating a conditional knockout. This system enables researchers to investigate a gene of interest in a tissue/cell (spatial control) and/or time (temporal control) specific manner.

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