Sato M, Saitoh I, Akasaka E, Inada E (2021) Development of a novel pipette tip-aided cell cloning method for the effective isolation of genome-edited porcine cell. OBM Genetics 5(1):16. doi: 10.21926/obm.genet.2101126

Summary: Isolation of clonal cells from a single colony is an essential step in the process of obtaining pure populations of stably-transfected clones after gene transfer and the subsequent drug selection. In the present study, a novel, simple, and non-invasive technique for the isolation of cells from single colonies using a disposable pipette tip was developed.

Usage: A toxin-based, drug-free selection system involving IB4-SAP was employed in the present study.

Related Products: IB4-SAP (Cat. #IT-10)

Galvan MA, Shramm PA, Bouajram R, Lappi DA, Ancheta LR (2019) A high efficacy selection method for transfected cells utilizing recombinant isolectin B4-saporin. Neuroscience 2019 Abstracts 794.10. Society for Neuroscience, Chicago, IL.

Summary: Transfection protocols often rely on the use of antibiotics for the selection of transfected cells and has become the accepted approach for in vitro research and therapeutic applications. Antibiotics have several shortcomings such as cost, continuous use, and harmful effects — even on the transfected cell population. In addition, selection pressures are often inefficient and fail to provide a population of cells that express the gene of interest (GOI) at high levels. We have used three separate GOI’s to select for solely high-expressing transfectants using targeted toxin selection pressure. Normal Rat Kidney Cells (KNRK) were individually transfected to express green fluorescent protein (GFP), melanopsin or the low-affinity nerve growth factor receptor (p75) using an innovative new transfection delivery vector called pGEI. The results from various assays were utilized to visually determine the expression rate and pattern of the targeted toxin selection method. Melanopsin and p75 — a photopigment and nerve growth factor, respectively — were of great interest to express in our transfected cells as a means to study their role in the development and function of neurons. The delivery vector, pGEI, removes resident Galalpha(1-3)Gal epitopes from non- human mammalian cell surfaces. This residue is the target of recombinant Isolectin B4 – Saporin (IB4-SAP), a selective targeted toxin. IB4-SAP is extremely potent, with an EC50 in the low picomolar range for alpha-D-galactopyranoside expressing cells in vitro. The cells with the highest expression of the inserted vector, and therefore the GOI, will have these residues removed. Those that fail to express the vector or do not express the vector in high enough amounts, will not have all the residues removed, and will be targeted and eliminated via IB4-SAP. This method of selection provides a means of purifying the highest- expressing transfected populations using a more cost-effective and time-saving approach.

Related Products: IB4-SAP (Cat. #IT-10)

Walker JR, Ong A, Detloff MR (2019) Role of nociceptive afferent input on forelimb reaching and grasping behaviors in the spinal cord injured rat. Neuroscience 2019 Abstracts 572.09. Society for Neuroscience, Chicago, IL.

Summary: Individuals with spinal cord injury (SCI) suffer a loss of motor and sensory function. The current standard of care to recover fine motor control is rehabilitation focused on a combination of range of motion, aerobic, and strength training (ST). However, limited research has been conducted to determine the role of nociceptive afferent inputs from muscle on spinal plasticity and/or recovery of function. Using a rodent model of SCI strength training rehabilitation, we determined that motor training not only improves forelimb strength and fine motor function but also can modulate the development of neuropathic pain, suggesting that improvements in reaching and grasping may be due, in part, to plasticity of nociceptive afferents. To further explore this, Sprague-Dawley rats received injections of rIB4-conjugated saporin, mu p75-conjugated saporin or unconjugated (vehicle) into the cervical dorsal root ganglia unilaterally to eliminate non-peptidergic and peptidergic nociceptors. There is an uninjured cohort and a group with unilateral C5 SCI. Von Frey and Hargreaves’ tests were performed at baseline and several time points post-injection to assess the effcacy of the nociceptive elimination. Several measures of forelimb strength were recorded over time including the isometric pull task, a single pellet retrieval task and the Montoya staircase test. To confirm the depletion of peptidergic and non-peptidergic nociceptors following saporin injection and/or SCI, cervical DRGs and spinal cords were stained with antibodies against CGRP and isolectin-B4. An understanding of the role of nociceptors in spinal plasticity and functional motor and sensory recovery of SCI patients will guide future research and refine rehabilitation strategies to further improve their quality of life.

Related Products: IB4-SAP (Cat. #IT-10), mu p75-SAP (Cat. #IT-16)

Araldi D, Bogen O, Green PG, Levine JD (2019) Role of nociceptor toll-like receptor 4 (TLR4) in opioid-induced hyperalgesia and hyperalgesic priming. J Neurosci 39(33):6414-6424. doi: 10.1523/JNEUROSCI.0966-19.2019

Objective: To evaluate the hypothesis that hyperalgesia and priming induced by opioids are mediated by similar nociceptor mechanisms.

Summary: Treatment with isolectin B4 (IB4)-saporin or SSP-saporin (which deplete IB4! and peptidergic nociceptors, respectively), or their combination, prevented systemic LDM-induced hyperalgesia, but not priming. HDM-induced priming, but not analgesia, was markedly attenuated in both saporin-treated groups.

Usage: IB4-SAP was diluted in saline and a dose of 3.2 μg in a volume of 20 μl administered intrathecally. SSP-SAP was diluted in saline and a dose of 100 ng in a volume of 20 μl was administered intrathecally.

Related Products: IB4-SAP (Cat. #IT-10), SSP-SAP (Cat. #IT-11)

Pinto LG, Souza GR, Kusuda R, Lopes AH, Sant’Anna MB, Cunha FQ, Ferreira SH, Cunha TM (2019) Non-peptidergic nociceptive neurons are essential for mechanical inflammatory hypersensitivity in mice. Mol Neurobiol 56(8):5715-5728. doi: 10.1007/s12035-019-1494-5

Objective: To determine the role of non-peptidergic nociceptors in mediating mechanical inflammatory hypersensitivity in mice.

Summary: rIB4-SAP oblates non-peptidergic neurons as displayed by the decrease of purinoceptor 3. The depletion of these neurons inhibited the mechanical inflammatory sensitivity induced by GDMF and carrageenan, but not by other expected factors such as nerve growth factor implying the role of this subset of neurons to be one of mediation as opposed to direct nociception.

Usage: IB4-SAP (0.16–3.2 μg/5 μl, i.t.), unconjugated Saporin (as control, 1.8 μg/5 μl, i.t.), or saline (vehicle, 5 μl/i.t.) were injected into the subarachnoid space on the midline between the L5 and L6 vertebrae.

Related Products: IB4-SAP (Cat. #IT-10), Saporin (Cat. #PR-01)

Watanabe S, Sakurai T, Nakamura S, Miyoshi K, Sato M (2018) The combinational use of CRISPR/Cas9 and targeted toxin technology enables efficient isolation of bi-allelic knockout non-human mammalian clones. Int J Mol Sci 19:E1075. doi: 10.3390/ijms19041075

Objective: Most genome editing systems employ transient treatment with selective drugs such as puromycin to obtain the desired genome-edited cells, which often allows some untransfected cells to survive and decreases the efficiency of generating genome-edited cells. The authors developed a novel targeted toxin-based drug-free selection system for the enrichment of genome-edited cells.

Summary: Results indicate that a combination of the CRISPR/Cas9 system and targeted toxin technology using IB4-SAP allows efficient enrichment of genome-edited clones, particularly bi-allelic KO clones.

Usage: Cells were trypsinized 3 days after transfection and approximately 80% were incubated for 30 min at 37°C in a solution (25 mcL) containing 0.5–1.0 mcg IB4-SAP (Cat. #IT-10).

Related Products: IB4-SAP (Cat. #IT-10)

Israel MR, Morgan M, Tay B, Deuis JR (2018) Toxins as tools: Fingerprinting neuronal pharmacology. Neurosci Lett 679:4-14. doi: 10.1016/j.neulet.2018.02.001

Summary: This review article provides an overview of the experimental techniques used to assess the effects that toxins have on neuronal function, as well as discussion on toxins that have been used as tools, with a focus on toxins that target voltage-gated and ligand-gated ion channels.

Related Products: IB4-SAP (Cat. #IT-10), NPY-SAP (Cat. #IT-28)

Araldi D, Khomula EV, Ferrari LF, Levine JD (2018) Fentanyl induces rapid onset hyperalgesic priming: Type I at peripheral and type II at central nociceptor terminals. J Neurosci 38(9):2226-2245. doi: 10.1523/JNEUROSCI.3476-17.2018

Objective: To evaluate priming, at both nociceptor terminals, the effect of local administration of agents that reverse type I (protein translation) or type II [combination of Src and mitogen-activated protein kinase (MAPK)] priming

Summary: Fentanyl, acting at the -opioid receptor (MOR), induces hyperalgesia and hyperalgesic priming at both the central and peripheral terminal of nociceptors and this is mediated by endoplasmic reticulum Ca2 signaling. Priming in the central terminal is type II, whereas that in the peripheral terminal is type I. Our findings may provide useful information for the design of drugs with improved therapeutic profiles, selectively disrupting individual MOR signaling pathways, to maintain an adequate long-lasting control of pain.

Usage: IB4-SAP was diluted in saline and a dose of 3.2 μg in a volume of 20 μl and administered intrathecally 14 d before experiments

Related Products: IB4-SAP (Cat. #IT-10)

Ferrari LF, Khomula EV, Araldi D, Levine JD (2018) CD44 signaling mediates high molecular weight hyaluronan-induced antihyperalgesia. J Neurosci 38(2):308-321. doi: 10.1523/JNEUROSCI.2695-17.2017

Objective: To study the role of the cognate hyaluronan receptor, CD44, signaling in anti-hyperalgesia induced by high molecular weight hyaluronan (HMWH).

Summary: These results demonstrate the central role of CD44 signaling in HMWH-induced anti-hyperalgesia, and establish it as a therapeutic target against inflammatory and neuropathic pain.

Usage: Both IB4-SAP and SSP-SAP were diluted in saline to doses previously shown to deplete nonpeptidergic (3.2 mcg/rat for IB4-SAP) and peptidergic (100 ng/rat for SSP-SAP) fibers. The toxins were administered intrathecally, in a volume of 20 mcl, 14 d before intradermal injection of LMWH on the dorsum of the hindpaw. Treatment with either conjugate, or a combination of the two, did not significantly affect mechanical nociceptive threshold.

Related Products: IB4-SAP (Cat. #IT-10), SSP-SAP (Cat. #IT-11), Anti-CD44-SAP (Cat. #IT-72)

Havelin J, Imbert I, Sukhtankar D, Remeniuk B, Pelletier I, Gentry J, Okun A, Tiutan T, Porreca F, King T (2017) Mediation of movement-induced breakthrough cancer pain by IB4-binding nociceptors in rats. J Neurosci 37:5111-5122.. doi: 10.1523/JNEUROSCI.1212-16.2017

Objective: To define a novel preclinical measure of movement-induced breakthrough pain (BTP) that is observed in the presence of morphine controlling ongoing pain.

Summary: Novel compounds targeting IB4-binding nociceptors may improve pain management for cancer pain patients and other patient populations suffering from BTP that is inadequately treated by currently available medications.

Usage: To determine the effect of eliminating input from IB4-binding fibers, separate groups of rats received spinal administration of IB4-SAP or the control, Blank-SAP (3.2 mcg/20 mcl saline) followed by a 10 mcl flush of saline. Movement of an air bubble placed between drug solution and saline was used to monitor progress of the injection.

Related Products: IB4-SAP (Cat. #IT-10), Blank-SAP (Cat. #IT-21)