Corante

About this Author
DBL%20Hendrix%20small.png College chemistry, 1983

Derek Lowe The 2002 Model

Dbl%20new%20portrait%20B%26W.png After 10 years of blogging. . .

Derek Lowe, an Arkansan by birth, got his BA from Hendrix College and his PhD in organic chemistry from Duke before spending time in Germany on a Humboldt Fellowship on his post-doc. He's worked for several major pharmaceutical companies since 1989 on drug discovery projects against schizophrenia, Alzheimer's, diabetes, osteoporosis and other diseases. To contact Derek email him directly: derekb.lowe@gmail.com Twitter: Dereklowe

Chemistry and Drug Data: Drugbank
Emolecules
ChemSpider
Chempedia Lab
Synthetic Pages
Organic Chemistry Portal
PubChem
Not Voodoo
DailyMed
Druglib
Clinicaltrials.gov

Chemistry and Pharma Blogs:
Org Prep Daily
The Haystack
Kilomentor
A New Merck, Reviewed
Liberal Arts Chemistry
Electron Pusher
All Things Metathesis
C&E News Blogs
Chemiotics II
Chemical Space
Noel O'Blog
In Vivo Blog
Terra Sigilatta
BBSRC/Douglas Kell
ChemBark
Realizations in Biostatistics
Chemjobber
Pharmalot
ChemSpider Blog
Pharmagossip
Med-Chemist
Organic Chem - Education & Industry
Pharma Strategy Blog
No Name No Slogan
Practical Fragments
SimBioSys
The Curious Wavefunction
Natural Product Man
Fragment Literature
Chemistry World Blog
Synthetic Nature
Chemistry Blog
Synthesizing Ideas
Business|Bytes|Genes|Molecules
Eye on FDA
Chemical Forums
Depth-First
Symyx Blog
Sceptical Chymist
Lamentations on Chemistry
Computational Organic Chemistry
Mining Drugs
Henry Rzepa


Science Blogs and News:
Bad Science
The Loom
Uncertain Principles
Fierce Biotech
Blogs for Industry
Omics! Omics!
Young Female Scientist
Notional Slurry
Nobel Intent
SciTech Daily
Science Blog
FuturePundit
Aetiology
Gene Expression (I)
Gene Expression (II)
Sciencebase
Pharyngula
Adventures in Ethics and Science
Transterrestrial Musings
Slashdot Science
Cosmic Variance
Biology News Net


Medical Blogs
DB's Medical Rants
Science-Based Medicine
GruntDoc
Respectful Insolence
Diabetes Mine


Economics and Business
Marginal Revolution
The Volokh Conspiracy
Knowledge Problem


Politics / Current Events
Virginia Postrel
Instapundit
Belmont Club
Mickey Kaus


Belles Lettres
Uncouth Reflections
Arts and Letters Daily
In the Pipeline: Don't miss Derek Lowe's excellent commentary on drug discovery and the pharma industry in general at In the Pipeline

In the Pipeline

« Evolving Enzymes: Let 'Em Rip | Main | Merck Cuts Coming? Or Not? »

August 1, 2013

Knockout Mice, In Detail

Email This Entry

Posted by Derek

Everyone in biomedical research is familiar with "knockout" mice, animals that have had a particular gene silenced during their development. This can be a powerful way of figuring out what that gene's product actually does, although there are always other factors at work. The biggest one is how other proteins and pathways can sometimes compensate for the loss, a process often doesn't have a chance to kick in when you come right into an adult animal and block a pathway through other means. In some other cases, a gene knockout turns out to be embryonic-lethal, but can be tolerated in an adult animal, once some key development pathway has run its course.

There have been a lot of knockout mice over the years. Targeted genetic studies have described functions for thousands of mouse genes. But when you think about it, there have surely been many of these whose phenotypes have not really been noticed or studied in the right amount of detail. Effects can be subtle, and there's an awful lot to look for. That's the motivation behind the Sanger Institute Mouse Genetics Project, who have a new paper out here. They're part of the even larger International Mouse Phenotyping Consortium, which is co-ordinating efforts like this across several sites.

Update: here's an overview of the work being done. For generating knockout animals, you have the International Knockout Mouse Consortium at an international level - the IKMC, mentioned above, is the phenotyping arm of the effort. In the US, the NIH-funded Knockout Mouse Project (KOMP) is a major effort, and in Europe you have the European Conditional Mouse Mutagenesis Program (EUCOMM), which has evolved into EUCOMMTOOLS. Then in Canada you have NorCOMM, and TIGM at Texas A&M.

I like the way that last link's abstract starts: "Nearly 10 years after the completion of the human genome project, and the report of a complete sequence of the mouse genome, it is salutary to reflect that we remain remarkably ignorant of the function of most genes in the mammalian genome." That's absolutely right, and these mouse efforts are an attempt to address that directly. The latest paper describes the viability of 489 mutants, and a more complete analysis of 250 of them - still only a tiny fraction of what's out there, but enough to give you a look behind the curtain.

29% of the mutants were lethal and 13% were subviable, producing only a fraction of the expected number of embryos. That's pretty much in line with earlier estimates, so that figure will probably hold up. As for fertility, a bit over 5% of the homozygous crosses were infertile - and in almost all cases, the trouble was in the males. (All the heterozygotes could produce offspring).

The full phenotypic analysis on the first 250 mutants is quite interesting (and can be found at the Sanger Mouse Portal site.. Most of these are genes with some known function, but 34 of them have not had anything assigned to them until now. These animals were assessed through blood chemistry, gene expression profiling, dietary and infectious disease challenges, behavioral tests, necropsy and histopathology, etc. Among the most common changes were body weight and fat/lean ratios (mostly on the underweight side), but there were many others. (That body weight observation is, in most cases, almost certainly not a primary effect. Reproductive and musculoskeletal defects were the most common categories that were likely to be front-line problems).

What stands out is that the unassigned genes seemed to produce noticeable phenotypic changes at the same rate as the known ones, and that even the studied genes turned up effects that hadn't been realized. As the paper says, these results "reveal our collective inability to predict phenotypes based on sequence or expression pattern alone." About 35% of the mutants (of all kinds) showed no detectable phenotypic changes, so these are either nonessential genes or had phenotypes that escaped the screens. The team looked at heterozygotes in cases where the homozygotes were lethal or nearly so (90 lines so far), and haploinsufficiency (problems due to only one working copy of a gene) was a common effect, seen in over 40% of those mutants.

Genes with some closely related paralog were found to be less likely to be essential, but those producing a protein known to be part of a protein complex were more likely to be so. Both of those results make sense. But a big question is how well these results will translate to understanding of human disease, and that's still an open issue. Clearly, many things will be directly applicable, but some care will be needed:

The data set reported here includes 59 orthologs of known human disease genes. We compared our data with human disease features described in OMIM. Approximately half (27) of these mutants exhibited phenotypes that were broadly consistent with the human phenotype. However, many additional phenotypes were detected in the mouse mutants suggesting additional features that might also occur in patients that have hitherto not been reported. Interestingly, a large proportion of genes underlying recessive disorders in humans are homozygous lethal in mice (17 of 37 genes), possibly because the human mutations are not as disruptive as the mouse alleles.

As this work goes on, we're going to learn a lot about mammalian genetics that has been hidden. The search for similar effects in humans will be going on simultaneously, informed by the mouse results. Doing all this is going to keep a lot of people busy for a long time - but understanding what comes out is going to be an even longer-term occupation. Something to look forward to!

Comments (14) + TrackBacks (0) | Category: Biological News


COMMENTS

1. qetzal on August 1, 2013 9:43 AM writes...

Isn't this almost exactly what Lexicon was initially doing, and selling access to the data to big pharma for target ID? I thought they had looked at a huge number of mouse genes.

Too bad their data can't be more readily accessed. I don't think they're even doing those kinds of pharma deals any more. I believe they're completely focused on developing their own drugs, so whatever they know from their knock-out studies that they haven't already licensed to someone else, they probably want to keep for themselves.

Permalink to Comment

2. bhip on August 1, 2013 10:41 AM writes...

Sadly, in my experience, assessments of knockout animals in drug discovery programs devolved into a box checking exercise in which negative data (i.e. data that raises suspicion on the drug target) could be explained away based on some of the valid points raised by Derek (embryonic lethality vs function in adults, mouse vs human biology, etc).
On the bright side, knockout mice can make dandy tools for testing antibody fidelity...

Permalink to Comment

3. Anon on August 1, 2013 11:09 AM writes...

I was going to write #1's comment almost verbatim. I think right now they have a couple potential partners they want to work with them on a diabetes drug.

Permalink to Comment

4. a. nonymaus on August 1, 2013 12:17 PM writes...

It's surprising to me that 35% of the investigated mutations show no effect given that there is likely to be a bias towards creating or studying mutants with mutations in genes of interest to the scientist/demiurge involved. Wonder what ENCODE would say that 35% of mutated DNA was doing?

Permalink to Comment

5. RKN on August 1, 2013 12:26 PM writes...

Salutary indeed.

What do you want to bet that repeating the same experiments in a different mammal would produce a whole new set of results, strikingly different than what was discovered in the mouse?

The feeling I have about the people doing this kind of research is analogous to how I feel about courageous troops fighting a pointless war.

Permalink to Comment

6. CR on August 1, 2013 12:50 PM writes...

Sanger isn't alone in their large-scale knockout generation and phenotyping efforts. The IKMC is a central hub for most of the efforts, including KOMP, EUCOMM, and others.

Permalink to Comment

7. Anonymous on August 1, 2013 1:22 PM writes...

Echoing #1 and #3, Lexicon has been generating and utilizing mouse knock-outs for well over a decade now.
http://lexpharma.com/research/drug-discovery/gene-knockouts.html

Permalink to Comment

8. luysii on August 2, 2013 5:37 AM writes...

"You can see a lot just by looking" Y. Berra

Permalink to Comment

9. Suleman on August 2, 2013 6:10 AM writes...

I used to know immunology researchers working in the knock-out field. They talked of how knocking out important cytokine genes would sometimes have little effect. The theory was that the immune system needed backup systems (redundancy) in case an infecting agent managed to sabotage a particular function. Clearly the immune system is a special case, but I wonder whether other systems also have such redundancy function, helping the mouse to make it to reproduction if something goes wrong with a key gene. If redundancy functions are important generally then it would very much complicate phenotype analysis.

Permalink to Comment

10. anonymous on August 2, 2013 6:50 AM writes...

Two (obvious?) points:
Knockout data is just another example of where, if Senior Mgmt LIKES your target, it doesn't matter which way the data goes (what, no phenotype? - just developmental compensation) and if they HATE your target, only the "negative" effects observed in the KO are relevant (its embryonic lethal - can't touch that one !)

Key to remember that many of the "best" drug targets are indeed embryonic lethal - e.g., HMG CoA reductase !

Permalink to Comment

11. anonymous on August 2, 2013 9:42 AM writes...

The drawbacks of knockout mice is well aware by the scientific community. Below is from the Knockout Mice web page of NHGRI (http://www.genome.gov/12514551#al-3)

While knockout mice technology represents a valuable research tool, some important limitations exist. About 15 percent of gene knockouts are developmentally lethal, which means that the genetically altered embryos cannot grow into adult mice. The lack of adult mice limits studies to embryonic development and often makes it more difficult to determine a gene's function in relation to human health. In some instances, the gene may serve a different function in adults than in developing embryos.

Knocking out a gene also may fail to produce an observable change in a mouse or may even produce different characteristics from those observed in humans in which the same gene is inactivated. For example, mutations in the p53 gene are associated with more than half of human cancers and often lead to tumors in a particular set of tissues. However, when the p53 gene is knocked out in mice, the animals develop tumors in a different array of tissues.

Despite these drawbacks, knockout mice offer one of the most powerful means now available for studying gene function in a living animal. Such studies will accelerate efforts to translate newfound knowledge of the human and mouse genomes into better strategies for diagnosing, treating and preventing human disease.

Permalink to Comment

12. Dr. Manhattan on August 4, 2013 8:12 PM writes...

An interesting alternative to gene knockouts is Forward Genetics, in which mice are mutagenized and the progeny are examined for interesting phenotypes. A good example and application is Bruce Beutler's work in immunology, using the mouse system. Here, rather than total gene ablation, point mutations and created at a higher rate and mice are screened for phenotypes of interest. Although he focuses on immune system phenotypes, in fact he gets all sorts of interesting phenotypes as a result. A description can be found here: Blood (2009) 113(7):1399-1407 and Nat Immunol. 2007 (7):659-664.

Permalink to Comment

13. Bernard Dwyer on August 7, 2013 7:25 AM writes...

I enjoy your posts, although, as I'm not a scientist, the enjoyment comes mostly from your style of writing in "things I won't work with". As a "barely made the pass mark in high-school chemistry" follower, I can just begin to comprehend the substance of your work, it's still fun to stimulate the grey matter with discussions just beyond my comprehension. Venturing into popular culture, the phrase "a gene knockout turns out to be embryonic-lethal," immediately brought a flashback to a scene in Blade Runner, where Tyrell tells the replicant Roy Batty that experiments in lengthening the lifespans of replicants created "a virus so lethal the subject was dead before it even left the table." Not remotely relevant to real and important research, I know, but isn't it interesting what connections we make? Thanks for your efforts, and thanks for an interesting read whenever I happen to trawl through my browser bookmarks. Cheers, Bernie Dwyer

Permalink to Comment

14. Big Freddie on August 12, 2013 3:14 PM writes...

Interesting post...that I am a bit late too...the Evo folks have been interested in the "no effect" knock out for many years...a notable idea is that orthologs can partially compensate for each other's loss, but depending on how much functional divergence there has been there is unlikely to be "perfect redundancy". As an Evo Devo person I have always been a little mystified reading pharma stuff as a consultant...where I will get interesting statements from chemists like "well, the eight proteins in the gene family are 75% similar at the amino acid level. Our drug has high specific activity and good energetics with target #5, which is associated with disease, can you tell us if it might interact with the other seven?"....My answer is...no...express the other seven and test them, and do mRNA insitus to see which members are co-expressed with #5 in your tissue of interest...neither of which they ever seemed to do to be honest...only to find that their drug has either no effect (a protein with a similar function is expressed in the same tissues during development and adulthood as #5, but has lower affinity for the drug, compensating for the chemical inhibition of the first target) or has broad "off target" effects because the drug binds to all members or the protein family which have evolved differential expression in many tissues. So..careful around the gene families folks...lots of evolution occurs due to neutral processes,positive/adaptive selection is not necessarily honing proteins into perfect targets for chemists...I wonder when pharma and evolution folks are going to forge a better relationship? Of course, with ENCODE running about "writing fiction" about evolution this might all be for naught...but it really surprises me that I visit pharma cos and never meet the molecular evolution section...seems like a staffing problem. Anyhoo, wikipedia ref for fun...or scariness :-)
http://en.wikipedia.org/wiki/Evolution_by_Gene_Duplication_(Theoretical_models)

Permalink to Comment

POST A COMMENT




Remember Me?



EMAIL THIS ENTRY TO A FRIEND

Email this entry to:

Your email address:

Message (optional):




RELATED ENTRIES
Gitcher SF5 Groups Right Here
Changing A Broken Science System
One and Done
The Latest Protein-Protein Compounds
Professor Fukuyama's Solvent Peaks
Novartis Gets Out of RNAi
Total Synthesis in Flow
Sweet Reason Lands On Its Face