Cover Article: Targeted Toxins – Cheaper and Quicker than Knockouts

Cell-specific targeting and removal: cheaper and quicker than knockouts with high impact results.

Knockout models are helpful tools in scientific research. They have been useful in studying and modeling in all sorts of research in biology; so much so that the pioneers – Smithies, Capecchi and Evans – won a well-deserved Nobel Prize for Physiology and Medicine in 2007. But there are some reasons not to go down that path:

1) About 15% of gene knockouts are developmentally lethal (from www.genome.gov).

2) According to information posted on a major university’s core facility website, it will take a minimum of 40 weeks to produce a knockout mouse. The cost for this best-case scenario is at least $11,000. And that doesn’t include the time and money spent for the molecular biology construction.

3) Statistics published by university core facilities range from 10% to 50% success rate in producing gene-based knockout models.

4) You are pretty much limited to mice. Of course, if you want to spend more, you can try rats.

That’s a lot of time and money for a grad student or post-doc to find out, “Gee, that didn’t give me a high impact result.”

Here’s an alternative that has a high rate of success, can give high impact results, takes about 10 days to see behavioral results, and costs $200 to $700 to treat about 20 mice. Or you can use rats. Or ferrets. Or many other species.

Targeted toxins offer the ability to develop “knockouts” through cell surface-based targeting that has several advantages over the gene-based approach. The “knockout” has a slight but important difference: instead of knocking out a particular protein from a set of cells (or even the whole animal), you eliminate a particular cell type. And this happens at your convenience: you inject the animal, put it back in its cage and then usually four days later, behavioral differences begin to show. These usually become permanent after a week or so. So you don’t have to wait 40 weeks to even start your experiments. People usually begin immunohistochemistry after a couple of weeks.

This figure illustrates how targeted toxins can be used to ‘knockout’ specific, cell surface-based targets.
Internalization of Substance P receptor (SPR) after injection of SP-SAP into the cerebrospinal fluid. This pseudo-color figure shows SPR in red after concentration due to internalization. Lesser concentrated SPR, and also that which is still on the surface membrane is shown in yellow.

The Cost

Let’s say you’re using a rat. Well, there’s the cost of the rat and its boarding. Often people use 100 ng of the targeted toxin. Since the price is usually $350 per 25 micrograms, for a single injection that would be $1.40 per animal. Then there’s the 2-week wait, the cost of the behavioral experiment, and the IHC. That’s less than $11,000. Much less. And then publish in Science, Nature, Journal of Neuroscience, Diabetes, Cancer Research, Endocrinology, Journal of Immunology, or many others.

The Animal Model

Using targeted toxins, you can begin with a fully mature, healthy animal. And the result is a fully mature, healthy animal, but with something missing which usually doesn’t affect its overall health and the animal is ready to run through mazes.

Now you know you’ve got a choice.

Select your Targeted Toxin now!

Douglas A. Lappi (2011) Targeting Trends 12(3):1,6.

Recent Publications using ATS Products

Borkowski LF, & Nichols NL. A2A and 5-HT Receptors are Differentially Required for Respiratory Plasticity Over the Course of Motor Neuron Loss in Intrapleurally CTB-SAP Treated Rats. (2019). FASEB J, 33 (1_supplement):843.843-843.843.

IT-14: CTB-SAP Dose: Bilateral, intrapleural injections of: 1) CTB-SAP (25 μg), or 2) un-conjugated CTB and SAP (control) in rats.

Toledo C, Andrade DC, & Del Rio R. Brainstem pre-sympathetic neurons contribute to irregular breathing patterns in volume overload heart failure. (2019). The FASEB Journal, 33 (1_supplement):lb630-lb630.

IT-03:  Anti-DBH-SAP   Dose:  Stereotaxic bilateral injections of Anti-DBH-SAP (5 ng/150 nl).

Sajjadi E, Seven YB, Simon AK, Zwick A, Satriotomo I, & Mitchell GS. Adenosine 2A Receptor Inhibition Promotes Neuroprotection Following Toxic Insult to Phrenic Motor Neurons. (2019). FASEB J, 33 (1_supplement):844.843-844.843.

IT-14: CTB-SAP Dose:  CTB-SAP selectively killed nearly all phrenic motor neurons within a week and caused diaphragm paralysis (p<0.01).

Souza G, Stornetta R, Stornetta D, Abbott S, & Guyenet P. Contribution of retrotrapezoid nucleus and carotid bodies to asphyxia-induced arousal in rats. (2019). FASEB J, 33 (1_supplement):733.736-733.736.

IT-11:  SSP-SAP Dose:  RTN was nearly completely destroyed with microinjections of SSP-SAP  (2.4 ng).

Research Tools for Parkinson’s Disease

A review of the tools for studying Parkinson’s Disease

These results show that an antibody to the extracellular domain of the dopamine transporter (DAT) can be used to target midbrain dopaminergic neurons and that Anti-DAT-Saporin may be useful for producing a lesion very similar to the naturally-occurring neural degeneration seen in Parkinson’s Disease (Wiley et al., 2003).

  • Koshy Cherian A, Kucinski A, Wu R, de Jong IEM, & Sarter MA-Ohoo. Co-treatment with rivastigmine and idalopirdine reduces the propensity for falls in a rat model of falls in Parkinson’s disease. LID – 10.1007/s00213-018-5150-y [doi]. (2019). Psychopharmacology (Berl), [Epub ahead of print] (1432-2072 (Electronic). IT-01: 192-IgG-SAP
  • Kucinski A, Kim Y, & Sarter M. Basal forebrain chemogenetic inhibition disrupts the superior complex movement control of goal-tracking rats. (2019). Behav Neurosci, 133 (1):121-134. 2019/01/29IT-01: 192-IgG-SAP
  • Murillo-Rodriguez E, Millan-Aldaco D, Palomero-Rivero M, Morales-Lara D, Mechoulam R, & Drucker-Colin R. Cannabidiol partially blocks the sleepiness in hypocretin-deficient rats. Preliminary data. (2019). CNS Neurol Disord Drug Targets2019/10/24.    IT-20:  Orexin-SAP
  • Dobryakova YV, Volobueva MN, Manolova AO, Medvedeva TM, Kvichansky AA, Gulyaeva NV, Markevich VA, Stepanichev MY, & Bolshakov AP. Cholinergic Deficit Induced by Central Administration of 192IgG-Saporin Is Associated with Activation of Microglia and Cell Loss in the Dorsal Hippocampus of Rats. (2019). Front Neurosci13 146. 2019/04/02. PMC6424051.    IT-01192-IgG-SAP
  • Ermine CM, Wright JL, Frausin S, Kauhausen JA, Parish CL, Stanic D, & Thompson LH. Modelling the dopamine and noradrenergic cell loss that occurs in Parkinson’s disease and the impact on hippocampal neurogenesis. (2018). Hippocampus, 28 (5):327-337. 2018/02/13. Pmc5969306.    IT-03: Anti-DBH-SAP
  • Oliveira LM, Falquetto B, Moreira TS, & Takakura AC. Orexinergic neurons are involved in the chemosensory control of breathing during the dark phase in a Parkinson’s disease model. (2018). Exp Neurobiol, 309 107-118.      IT-20: Orexin-SAP; IT-35: Rabbit IgG-SAP
  •       Oliveira LM, Moreira TS, & Takakura AC. Raphe Pallidus is Not Important to Central Chemoreception in a Rat Model of Parkinson’s Disease. (2018). Neuroscience, 369 350-362. 2017/12/02IT-23: Anti-SERT-SAP; PR-01: Saporin

More information:    

192-IgG-SAPAnti-DBH-SAP Anti-SERT-SAPOrexin-SAPAnti-DAT-SAP

Alzheimer’s Disease models

A review of the tools for creating animal models of Alzheimer’s Disease

“192 IgG-saporin binds selectively and irreversibly to low-affinity nerve growth factor receptor interrupting cholinergic neuronal protein synthesis.”

“Anti-DBH-SAP allows a selective and gradual lesioning of noradrenergic neurones in the brain stem nucleus locus coeruleus, the primary site of noradrenaline production in the CNS.”

Verkhratsky A, Parpura V, Rodriguez-Arellano J, & Zorec R. (2019). Astroglia in Alzheimer’s Disease. In A Verkhratsky, M Ho, R Zorec & V Parpura (Eds.), Neuroglia in Neurodegenerative Diseases (Vol. Advances in Experimental Medicine and Biology, vol 1175, pp. 273-324). Singapore: Springer.   

For more information:      IT-01:  192-IgG-SAP and IT-03:  Anti-DBH-SAP

Summary:  192-IgG-SAP binds selectively and irreversibly to low-affinity nerve growth factor receptor interrupting cholinergic neuronal protein synthesis was employed.  Anti-DBH-SAP binds dopamine-β-hydroxylase, which is not only localized mainly in the cytosol, but also at the plasma membrane surface of noradrenergic neurons.  Anti-DBH-SAP produced specific and dose-dependent depletions of locus coeruleus neurons, with no effects on other cholinergic, dopaminergic or serotonergic neuronal populations.  The possibility to induce a partial or total noradrenergic loss (by varying the injected dose) makes this immunotoxic approach an ideal model to study events within the noradrenergic projection system, as they occur during age-related demise of locus coeruleus in humans.

Award winner for animal model of temporal lobe epilepsy

Congratulations to Dr. Argyle Bumanglag and the team at University of Florida for the important work they presented using SSP-SAP to create an animal model of temporal lobe epilepsy.

Injections of SSP-SAP into the hippocampus of a rat causes acute hippocampal injury, permanent dentate granule cell-onset epilepsy, and hippocampal sclerosis that closely resembles the selective hippocampal pathology exhibited by patients diagnosed with TLE. The rats are chronically epileptic, with data going out a year for lesioned animals.

Also see the recent publication:

Chun E, Bumanglag AV, Burke SN, & Sloviter RS. Targeted hippocampal GABA neuron ablation by Stable Substance P–saporin causes hippocampal sclerosis and chronic epilepsy in rats. (2019). Epilepsia60 (5):e52-e57.

Objective: Hippocampal GABA neurons were targeted for selective elimination to determine whether a focal hippocampal GABAergic defect in an otherwise normal brain can initiate cryptogenic temporal lobe epilepsy with hippocampal sclerosis.
Summary:  Hippocampal GABAergic dysfunction is epileptogenic and can produce the defining features of cryptogenic temporal lobe epilepsy.
Dose:  Intrahippocampal injections of SSP-SAP (0.4 ng/10 nL) were performed using a 0.5-μL Neuros Syringe lowered into four hippocampal sites along both the transverse and longitudinal hippocampal axes bilaterally.

Abstracts from Society for Neuroscience (SFN) Symposium November 3-7, 2018 – San Diego, CA

049.05 / S3 Learning and memory improvement mediated by CB1 cannabinoid receptors in animal models of cholinergic dysfunction

M. MORENO-RODRÍGUEZ, J. MARTÍNEZ-GARDEAZABAL, A. LLORENTE-OVEJERO, L. LOMBARDERO, I. MANUEL, *R. RODRIGUEZ-PUERTAS

featuring IT-01 192-IgG-SAP

128.20 / M17 Screening targeting agents and their cell surface biomarkers for high specificity and rapid internalization via cell death and fluorescence

*L. ANCHETA1, R. BOUAJRAM2, D. A. LAPPI3;

featuring IT-27 Streptavidin-ZAP

174.27 / JJJ31 Improvements of cognitive function by focused ultrasound associated with adult hippocampal neurogenesis in immunotoxin 192-Saporin rat model of cholinergic degeneration

*C. KONG1, J. SHIN1,2, J. LEE1,2, C. KOH1, J. SIM1,2, Y. NA3, W. CHANG1, J. CHANG1,2

featuring IT-01 192-IgG-SAP

238.14 / ZZ15 Dissociable effects of Noradrenergic and Cholinergic lesions of Anterior Cingulate Cortex on distractibility

*J. A. MCGAUGHY1, D. J. HUTCHINS2, A. J.2, C. S. PIMENTEL2, J. A. SWAINE2

featuring IT-03 Anti-DBH-SAP

325.09 / DDD22 Noradrenergic modulation of the orbitofrontal cortex mediates flexibility of goal-directed behavior

*J.-C. CERPA1,2, A. R. MARCHAND1,2, M. WOLFF1,2, S. L. PARKES1,2, E. COUTUREAU1,2

featuring IT-03 Anti-DBH-SAP

379.29 / L1 Sonic hedgehog signalling pathway during regenerative processes in a mouse model of spinal motoneuronal loss

*M. GULISANO1, N. VICARIO2, A. COSTANTINO3, M. A. S. GIUNTA2, F. M. SPITALE2, R. PARENTI2, R. GULINO3

featuring IT-14 CTB-SAP

491.01 / MM13 Impaired reach-to-grasp responses in mice depleted of striatal cholinergic interneurons

*N. ABUDUKEYOUMU, M. GARCIA-MUNOZ, Y. NAKANO, G. W. ARBUTHNOTT

featuring IT-42 Anti-ChAT-SAP

512.05 / GGG8 Lifespan and cholinergic changes in cognitive flexibility in rats

*C. CAMMARATA1, E. D. DE ROSA2, A. K. ANDERSON3

featuring IT-01 192-IgG-SAP

679.23 / VV4 SUVN-G3031, H3 receptor inverse agonist produces wake promoting activity in rats with hypocretin-2-saporin lesions of the lateral hypothalamus

*S. DARIPELLI, G. BHAYRAPUNENI, C. TIRUMALASETTY, V. BENADE, R. SUBRAMANIAN, S. PETLU, N. PRAVEENA, P. JAYARAJAN, A. SHINDE, R. BADANGE, V. BHATTA, R. NIROGI

featuring IT-20 Orexin-SAP

761.02 / MM11 Exercise is neuroprotective following partial motoneuron depletion: Run for your dendrites

*C. CHEW1, D. R. SENGELAUB2

featuring IT-14 CTB-SAP

773.20 / YY14 Evidence that the LH surge in ewes involves both neurokinin B-dependent and -independent actions of kisspeptin

*R. L. GOODMAN1, J. A. LOPEZ1, M. N. BEDENBAUGH1, J. M. CONNORS1, S. L. HARDY1, S. M. HILEMAN1, L. M. COOLEN2, M. N. LEHMAN3

featuring IT-63 NK3-SAP; IT-21 Blank-SAP; Custom Conjugate

Abstracts from Society for Neuroscience (SFN) Symposium October 19-23, 2019 – Chicago, IL

048.01 / F11 – Increased transplantation efficacy of mesenchymal stem cell by focused ultrasound and improvement of the spatial memory in the 192 IgG-saporin rat model

*J. LEE1,2, Y. SEO1,2, J. SHIN1,2, C. KONG1, Y. NA3, W. CHANG1, J. CHANG1,2;

featuring IT-01 192-IgG-SAP

052.10 / H30 – Nociceptors expressing TRPV1 and trigeminal nucleus neurons expressing NK1 mediate orthodontic pain

*S. WANG1, M. KIM1, K. ONG1, E.-K. PAE2, M.-K. CHUNG1;

featuring IT-11 SSP-SAP

079.08 An acetylcholine-dopamine interaction in the rat nucleus accumbens and its tentative involvement in ethanol’s dopamine-liberating effect

A Andrén,, L Adermark, B Söderpalm,, M Ericson

featuring IT-42 Anti-ChAT-SAP

134.13 / H15 – Exercise is neuroprotective following partial motoneuron depletion via androgen action at the target muscle

*C. CHEW, D. R. SENGELAUB;

featuring IT-14 CTB-SAP

158.03 / V8 – Targeted hippocampal GABA neuron ablation produces hippocampal sclerosis, epilepsy, and dissociable effects on the Morris water maze and object-place paired association tasks

*L. M. TRUCKENBROD1, A. V. BUMANGLAG4, E. CHUN5, A. HERNANDEZ6, Q. P. FEDERICO2, A. P. MAURER3, R. S. SLOVITER5, S. N. BURKE2;

featuring IT-11 SSP-SAP

218.22 / H28 – Maintenance mechanism of nociplastic pain in males

*K. E. MCDONOUGH1, K. M. HANKERD2, J.-H. LA2, J. M. CHUNG3;

featuring IT-06 Anti-Mac-1-SAP mouse

331.10 / AA12 – Sign-trackers deploy perceptual, but not cholinergic-attentional, mechanisms to respond to salient cues

*K. B. PHILLIPS, C. AVILA, M. SARTER;

featuring IT-01 192-IgG-SAP

336.01 / BB54 – Effect of medial septal selective and non selective lesions on exploratory behavior and recognition memory

*L. KRUASHVILI, G. BESELIA, N. CHKHIKVISHVILI;

featuring IT-01 192-IgG-SAP

377.10 / D25 – How to stimulate: Basal forebrain DBS parameters to restore the attentional performance of rats with cholinergic losses

*M. NAZMUDDIN1, H. A. RAO2, T. VAN LAAR1, M. F. SARTER2;

featuring IT-01 192-IgG-SAP

418.06 / Y37 – Effects of an orexin-2 receptor agonist on attention in rats following loss of cortical cholinergic projections

*S. A. BLUMENTHAL1, E. B.-L. MANESS2, J. R. FADEL3, J. A. BURK1;

featuring IT-01 192-IgG-SAP

418.11 / Y42 – Dissociable attentional effects of dopaminergic and cholinergic lesions to the anterior cingulate cortex

*M. K. CLEMENT, C. S. PIMENTEL, J. A. SWAINE, A. J. PIMENTEL, D. HUTCHINS, J. A. MCGAUGHY;

featuring IT-01 192-IgG-SAP

502.07 / U40 – SUVN-G3031, histamine H3 receptor inverse agonist preclinical evaluation for the treatment of excessive daytime sleepiness in narcolepsy

*G. BHYRAPUNENI, V. BENADE, S. DARIPELLI, V. KAMUJU, A. SHINDE, R. ABRAHAM, R. NIROGI, V. JASTI;

featuring IT-20 Orexin-SAP

572.09 / H38 – Role of nociceptive afferent input on forelimb reaching and grasping behaviors in the spinal cord injured rat

*J. R. WALKER, A. ONG, M. R. DETLOFF;

featuring IT-10 IB4-SAP; IT-16 mu p75-SAP

591.04 / S10 – Leptin receptor activity in the nucleus of the solitary tract increases forebrain leptin sensitivity

*R. B. HARRIS;

featuring IT-47 Leptin-SAP

601.19 / Y37 – Medial septum cholinergic signaling regulates gastrointestinal-derived vagus sensory nerve communication to the hippocampus

*A. N. SUAREZ1, C. M. LIU2, A. M. CORTELLA1, E. N. NOBLE1, S. E. KANOSKI1;

featuring IT-01 192-IgG-SAP; IT-31 CCK-SAP

614.03 / DD15 – In vivo monitoring of cholinergic neurotransmission with a microelectrochemical choline biosensor

S. DOYLE1, M. M. DORAN1, K. L. BAKER1, C. CUNNINGHAM2, *J. P. LOWRY1;

featuring IT-16 mu p75-SAP

786.03 / AA41 – The role of subcortical hippocampal inputs in contextual memory formation

*V. S. GRAYSON1, Y. HAN3, A. L. GUEDEA1, V. JOVASEVIC1, C. GAO4, A. APKARIAN2, J. M. RADULOVIC1;

featuring IT-01 192-IgG-SAP

789.11 / BB38 – Selective loss of septohippocampal cholinergic projections is associated with more circuitous homeward progressions

*J. R. OSTERLUND1, A. A. BLACKWELL1, M. LIPTON1, V. CASTILLO1, G. L. KARTJE2, S.-Y. TSAI3, D. G. WALLACE1;

featuring IT-01 192-IgG-SAP

794.10 / CC54 – A high efficacy selection method for transfected cells utilizing recombinant isolectin B4-saporin

M. A. GALVAN, P. A. SHRAMM, R. BOUAJRAM, D. A. LAPPI, *L. R. ANCHETA;

featuring IT-10 IB4-SAP

SfN Poster of the Year Contenders

The Society for Neuroscience meeting is just around the corner.  Come visit us in Chicago  – October 19-23, Booth #763. 

Here is a list of the posters competing for the Annual Post of the Year Award using ATS products. (click to zoom)

Saturday, October 19

Sunday, October 20

Monday, October 21

Tuesday, October 22

Wednesday, October 23