Laser ablation is one of the earliest approaches used for killing cells of interest. The intense light produced by a laser is absorbed within a short time period and converted into heat, which damages cellular proteins and other macromolecules and leads to cell death within seconds¹¹. Laser pulses can damage the biological material on different scales. The power, repetition rate, and duration of the pulses will determine the extent of the damage¹². The main advantage of laser-mediated cell ablation over the chemical and genetic cell ablation methods described below is flexibility; a laser can eliminate every kind of cell in any physiological state¹³. Laser ablation allows for a precise control over the time of ablation and the ability to ablate cells at any stage of development, which are less feasible with chemical or genetic cell ablation^{12, 14} The technique has been employed extensively in developmental model systems like C. elegans and zebrafish¹⁵¹⁶¹⁷ However, it has its limitations; it is time consuming, labor-intensive, and requires expensive equipment¹². Because it needs to be combined with microscopic techniques, only targeted cell groups that can be visualized by microscopy are amenable to ablation ¹⁸ Development of vital fluorescent imaging systems in the past two decades has increased its efficiency and versatility^{18, 19} The most significant and obvious limitation of laser ablation is unavoidable damage to adjacent cells due to cytoplasmic boiling and gas bubbles generated by the high energy laser power¹². Second, ablating multiple cells in an individual animal is a tedious, time-consuming and labor-intensive task. Third, ablation of multiple cells can be inefficient since there are significant differences of laser light absorbance levels among cell types¹², and ablation of cells in deep locations requires higher levels of laser power than superficially-located cells ¹² For these reasons, laser ablation has been seldom applied to studying cell function in adult animals, but is still utilized for addressing fundamental questions in early development and in in vitro organ culture ^{20, 21}.

Optogenetic or photo-inducible cell ablation has been developed recently by combining genetic and laser ablation methods (Fig. 1) ¹¹. This technique often uses genetically encoded photosensitizers, which produce reactive oxygen species (ROS) upon light excitation (Fig. 1A and 1B) ^11,22 Photosensitizers, which include a red fluorescent KillerRed (Fig. 1A) ²³²⁴ and a green fluorescent mini singlet oxygen generator (miniSOG) (Fig. 1B)²⁵, transmit energy from the absorbed green or blue lights to activate molecules in the acute cell necrosis¹¹. Precise photo-inducible ablation of cells such as neurons in vivo can also be achieved through cell-specific expression of a light-activated caspase-3, engineered by exploiting its spring-loaded activation mechanism through insertion of the light-sensitive protein (LOV2) domain that expands upon blue light exposure (Fig. 1C)²⁶. Optogenetic cell ablation methods are effective at single-cell resolution, with precise temporal control ¹¹, and have minimal off-target/non-specific cell death since they utilize a lower intensity of light than the laser ablation method. These optogenetic methods allow for selective ablation of cells in a temporally and spatially precise manner, facilitating the study of cell function in different tissues and developmental stages in various model systems, including vertebrates. However, the ability to photo-ablate cells is also limited by the accessibility and transparency of tissues for focused illumination of a region of interest. Optogenetics can be used for cell ablation by combining genetics and light stimulation, enabling the execution of well-defined events within genetically defined populations of cells, with precise temporal and spatial resolution.

Optogenetic approaches can be used for manipulating neuronal excitability transiently, which is an efficient way to probe causal relationships between specific neuronal cells and behavior. Chemogenetic tools have also been developed for this purpose. Both techniques have been widely used in the central nervous system (CNS) and peripheral sensory ganglia to manipulate neuronal activity in a cell-type-specific fashion both in vitro and in vivo, to determine functions of specific neuronal populations^27–32. Although these tools do not ablate neurons, they can rapidly silence them. Therefore, we will briefly discuss these two methods.

Some cell populations are especially vulnerable to specific chemical agents, which therefore can be used as toxins to target and eliminate those cells. We will briefly discuss some of these tools in the context of some of the diseases and/or cell types in which they have been applied.

Cre-mediated recombination of molecularly engineered loxP sites in animals has been widely used to generate a variety of targeted genomic rearrangements. Cre-mediated recombination with inverted loxP sites, which causes chromosomal loss and triggers apoptosis, is known as the targeted recombination between inverted loxP sites (TRIP) method of cell ablation⁵⁹. The targeted cell population is defined by both transgenic Cre-expression and the knock-in of inverted loxP sites in an animal’s genome. TRIP-mediated cell ablation is useful to investigate the function of a large variety of proliferating cell populations in a whole organism⁵⁹. Although this technique is promising, it has not yet been used widely. Limitations include: 1) its ability to only target proliferating cell populations, and 2) the potential for lethality if Cre is expressed constitutively or leaky in early stages of development.

Apoptosis is a morphologically distinct form of programmed cell death. It occurs in a wide range of mammalian cell types and plays important roles in both physiological and pathological conditions⁶⁰. Since apoptosis is an intrinsic process involving controlled dismantling of intracellular components while avoiding inflammation and damage to surrounding cells, manipulating it by targeting its central components, caspases, can provide an ideal approach to induce “suicide” of target cells with few bystander effects⁶¹. Caspases that are initially synthesized as inactive monomeric zymogens (pro-caspases) induce cell apoptosis after the pro-caspases are activated through dimerization and often oligomerization⁶². Taking advantage of cell killing by pro-caspase dimerization, several groups constructed pro-caspases fused to FK506-binding protein (FKBP), a chemically induced dimerization (CID)-binding site. Addition of FK506 (or its homodimer, FK1012), a nontoxic and lipid-permeable dimerizer, then aggregates and activates the pro-caspases ^{61, 63} Several groups have successfully used this method to deplete hepatocytes⁶⁴, adipocytes⁶⁵, pancreatic β-cells⁶⁶, retinal ganglion cells⁶⁷, and mixed populations of specific cell types⁶⁸. However, this tool can be affected by antiapoptotic factors in vivo. For example, adiponectin decreases caspase-8-mediated death of cardiac myocytes and pancreatic β-cells, whereas genetic ablation of adiponectin enhances caspase-8-induced apoptosis in vivo⁶⁹. FK506 is also a potent immunosuppressive agent with significant nephrotoxic properties⁷⁰. Finally, FKBPs are involved in regulating a wide range of biological and pathological processes in mammals without the binding of FK506⁷¹. These intrinsic features have the potential to confound experimental results.

Antibody (Ab)-mediated cell ablation based on targeting specific antigens expressed on immune cells has been used to deplete cells, including neutrophils, B cells, and T cells, in mice. The most commonly used antibody for neutrophil ablation is anti-Ly6G mAb, clone 1A8. In contrast to clone RB6–8C5, which cross-reacts with Ly6C, a receptor also present on other myeloid cells and lymphocytes^{72, 73}, clone 1A8 specifically recognizes and depletes Ly6G+ neutrophils, but not other cells⁷⁴. Anti-ly6G-mediated depletion of neutrophils in vivo depends on the function of macrophages but not the activation of complement ⁷⁵. Therapeutic B cell depletion by targeting CD20 has significantly improved treatment outcomes of non-Hodgkin’s lymphoma in humans⁷⁶. Anti-mouse CD20 mAb is also widely used to eliminate B cells in mice⁷⁷; a single dose depletes >95% of mature B cells in circulation and tissue by monocyte-mediated cellular cytotoxicity^{77, 78} Like B cells, CD4+ and CD8+ T cells can be eliminated in vivo by injection of CD4 or CD8 mAb, respectively, but the mechanisms responsible remain unknown^{79, 80} The available data indicates that, although antibody-mediated ablation is efficient, only cells expressing specific surface markers can be targeted. This strategy therefore cannot be applied to most myeloid cells, including dendritic cells and monocytes, because they lack appropriate antigens, and it is also limited by the availability of specific antibodies. Furthermore, because different antibodies kill cells by different mechanisms, it can be problematic to compare the functions of two distinct cell populations based on LOF studies with two different antibodies.

Application of the HSV-TK method to study the function of targeted cell populations evolved from anti-cancer therapy using the suicide gene HSV-TK⁸¹. HSV-TK phosphorylates nucleoside analogs into nucleotides. It phosphorylates the human nucleoside analog, ganciclovir (GCV), to form deoxythymidine triphosphate⁸², which is incorporated into the genome and causes cell death by inhibiting DNA synthesis only in proliferative cells (Fig. 2) ⁸³.HSV-TK cell ablation is achieved by oral administration of GCV to an animal, usually mice, engineered to express HSV-1-TK in a specific cell population (Fig. 2) ^{83, 84} This strategy will eliminate only cells that actively synthesize DNA and can therefore be used to ablate dividing, but not quiescent, cells. Also, this strategy has following limitations: 1) the toxic side effects of GCV on bone marrow cells; and 2) the off target effect on adjacent cells through transporting the metabolite triphosphate to nearby cells⁸⁵ (Fig. 2).

Bacterial nitroreductase (NTR)-induced cell ablation kills both actively dividing cells and nondividing cells. Bacterial NTR can activate the pro-drug CB1954 (5-aziridin-1 -yl-2–4-dinitrobezamide) into two DNA-damaging, cytotoxic metabolites, 2-hydroxylamine and 4- hydroxylamine⁸⁶ that can kill dividing and non-dividing cells⁸⁷. Various NTRs are found in nature. The most commonly used NTR is obtained from E. coli⁸⁷ NTR/prodrug CB1954-mediated cell ablation uses a transgenic strategy to express NTR in a specific cell population with administration of the pro-drug Metronidazole and CB1954⁸⁸ This method has been used to ablate post-mitotic cells such as neurons, astrocytes and adipocytes^{89- 91}. Its main limitations come from the effects of the toxic metabolites⁸⁸. 2-hydroxylamine can cause unavoidable bystander effects by entering adjacent non-targeted cell populations through gap junctions ^{92, 93}

Diphtheria toxin (DT) is a 535 amino acid polyprotein that is cleaved into to two fragments, A and B ¹. The A fragment is the amino terminal 190 amino acids containing the catalytically active toxin domain, whereas the B fragment (345 amino acids) contains the domain interacting with the cell surface receptor for DT. DT-mediated cell ablation model is generated by either transgenic expression of the DT-receptor (DTR) in the targeted cell population coupled with injection of DT to the animal (DT/DTR-mediated cell ablation) or by expressing active A fragment within cell populations ^{2, 94}, in the absence of the B fragment (DTA expressing cell ablation) (Fig. 3). Mice are not normally sensitive to DT since the Epithelial Growth Factor Receptor in mice does not bind DT as it does in other species ¹. In susceptible hosts, B fragment binding to the receptor causes the internalization and cleavage of A fragment (Fig. 3A)¹. The A fragment is internalized and binds to protein synthesis machinery, inhibiting protein synthesis leading to cell death via apoptotic pathway¹. Because the B fragment is inactive in rodents, researchers have primarily used the selective expression of DT-A chain to accomplish cell ablation in mice(Fig. 3A). Although the DT/DTR cell ablation model has been used to study cellular functionalities in vivo for more than 30 years^{1, 2}, it has limitations. It has a narrow pharmacological window, which prohibits dose dependent studies. This narrow range is primarily due to the extreme toxicity of DT, where even a single molecule may kill a cell⁹⁵. Several groups have recently reported that DT administration of only 2- to 3-fold higher doses than the effective dose results in significant off-target effects, including local lung and renal toxicity and significant weight loss, resulting in morbidity and mortality independent of the DTR⁹⁶⁹⁷ The narrow pharmacological window and off-target effects of the DT-mediated cell-ablation model often make it difficult to distinguish target dependent effects from off-target effects upon DT delivery in DTR transgenic mice. This method cannot be used in intermediate- to large-sized animals such as rabbits, sheep, or pigs, because in these animals, endogenous DTR binds to DT, causing apoptosis independently of transgene expression^{1, 2, 98} These facts underscore an unmet need to develop a new model that specifically ablates cells in vivo with higher efficiency and fewer off-target effects.

Transgenic expression of the diphtheria toxin fragment A (DT-A) subunit driven by a cell specific promoter is another useful cell ablation method (Fig. 3B)²⁹⁴ Since DT-A directly inhibits protein synthesis, DT-A within a cell will lead to cell death via apoptotic pathway, and no further activation steps are needed(Fig. 3B). This makes DT-A an attractive tool for the specific ablation of cells. The DT-A gene used for eliminating cells is engineered so that the protein product will be retained within the cytoplasm. In the absence of DT-B, any DT-A released from dying cells does not affect neighboring cells. By this method, groups have successfully killed pancreatic cells (Fig. 3B)⁹⁹, growth hormone-expressing cells¹⁰⁰ and brown adipose tissue¹⁰¹. However, this method easily causes lethality when the promoter driving DT-A is active in multiple cell types or in cell types that are essential for embryo viability¹⁰². In addition, because of the extreme toxicity of DT-A, lethality or side effects may occur if the so-called tissue- or cell-specific promoter has any ectopic DT-A gene expression outside the intended lineage¹⁰³. In order to improve this method, researchers have tried to generate attenuated DT-A. Attenuated DT-A requires a substantially higher level of expression than its nonattenuated form to produce a lethal event ¹⁰⁴ Generation of the conditional expression of DT-A mice employing Cre recombinase and Cre/ER system can tightly control the expression of DT-A in target cells at specific life stage (Fig. 3B)^{103, 105–107} By genetic modulation, DT-A mediated cell ablation has been successfully applied in depleting T cells¹⁰⁸, retinal pigment epithelium¹⁰⁷ and myogenic cell populations¹⁰⁹.

Except for the laser-mediated technique, all of the methods so far discussed produce cell ablation that occurs relatively slowly, from several hours up to a day¹¹⁰. This is a disadvantage when studying relatively rapid cellular events occurring over a time course of seconds to minutes during development or normal physiological function. We next describe a novel method for mediating rapid cell ablation.

We recently created a new method of conditional and rapid cell ablation⁹⁸. This model is based on findings that intermedilysin (ILY)¹¹¹, exclusively lyses human cells but not cells from any other species through binding to the human complement regulatory protein CD59 (hCD59). ILY is a cholesterol-dependent cytolysin that is secreted by Streptococcus intermedius. ILY specifically lyse human cells by binding to hCD59 and forming a pore (Fig. 4). The C-terminus of ILY domain 4 (D4) encounters the membrane first^{112, 113} The binding of D4 triggers a structural rearrangement in which ILY monomers oligomerize and form a pre-pore complex that causes lysis of the cells¹¹². ILY oligomerizes with as many as 50 other ILY molecules to form large diameter pores^{112, 113} through which ILY rapidly lyses the cells by necrosis (Fig. 4) ¹¹² Interestingly, ILY does not lyse CD59 positive (CD59+) cells from 9 other animal species (cat, chicken, cow, dog, horse, rabbit, sheep, rat, and mouse) that were tested because it does not bind to the CD59 protein of these species^{111, 112} Additionally, we have documented that human serum contains ILY-specific neutralizing antibodies not found in any other animal species⁹⁸. Another exciting characteristic of ILY is its large pharmacological window⁹⁸. No cell lysis was observed even when the ILY concentration was 5,000-fold greater than the dose that is required to lyse 50% of human erythrocytes⁹⁸.

We have taken advantage of ILY’s unique binding to hCD59 as well as the absence of the neutralizing Ab against ILY in any other animal species and developed an ILY- mediated hCD59-expressing cell ablation model in mice ⁹⁸ (Fig. 4). Recently, we also confirmed its effectiveness by generating hCD59 transgenic rats that specifically express hCD59 only on the surface of their red blood cells under the control of the hemoglobin promoter^{54, 98} CD59 is a glycosylphosphatidylinositol-linked (GPI-linked) membrane protein that inhibits formation of the membrane attack complex (MAC) in the complement system by binding to complement (C) proteins C8 and C9, preventing C9 incorporation and polymerization¹¹⁴. Over the past fifteen years, our group has focused on generating CD59 knockout^115–118 and hCD59 transgenic mice to investigate the role of CD59 in the pathogenesis of human diseases^119–122. Importantly, over-expression of CD59 does not cause any abnormalities in vitro or in vivo^{98, 116}, ^122–127

To increase the usefulness of ILY/hCD59 cell ablation, we have explored the potentially broad application of the ILY-mediated cell-ablation model by generating Cre- inducible hCD59 transgenic mice (Fig. 4) ¹²⁸ Specifically, we generated a line of Cre- inducible floxed STOP-hCD59 transgenic mice, where specific hCD59 expression occurs following Cre-mediated recombination (ihCD59). By crossing ihCD59 transgenic mice with transgenic mice that express Cre in a cell-specific manner or after delivery of an adenovirus expressing Cre, we obtained several lines that specifically expressed ihCD59 in a spatially regulated manner on the surface of immune, epithelial, or neuronal cells¹²⁸. ILY injection resulted in conditionally specific ablation of various types of cells without detectable off-target effects, including on evidence of lysis to cells in adjacent tissues ¹²⁸ Moreover, we tested this ablation technique in diverse disease models and found it valuable for the study of cellular functions, regeneration, and neuronal and tissue injury¹²⁸.

Together, in the section, we have reviewed versatile tools for ablating the targeted cell populations including the neuronal cells and discussed their features (Table 1). In the next, we will discuss how to apply these tools for the studies.

Dissecting normal cell function and normal cellular interactions is fundamental to understanding multi-cellular life. Thorough characterization of a targeted cell population’s function under physiological conditions also facilitates understanding of its pathogenic role and of how to cause it to regenerate after ablation. The LOF approach is essential for achieving these goals.

Cell ablation tools are also powerful for studying the role of the microenvironment in tumorigenesis. Tumorigenesis, also called oncogenesis is the process that transforms normal cells into malignant cancer cells. This intricate process is associated with cellular, genetic, and epigenetic changes and abnormal cell division¹⁴⁶. The role of the microenvironment in tumor survival, progression, metastasis, immune evasion, and therapy resistance is only recently beginning to be better appreciated. Tumors are complex cellular structures that incorporate cancer cells, host cells of various lineages, and stroma. Host cells in tumors include endothelial cells that form blood and lymphatic vessels, tumor-associated macrophages (TAMs), fibroblasts, circulating blood cells, extravasated white blood cells, and platelets. The microenvironment is essential for tumor establishment, growth, infiltration of neighboring tissues, immune evasion, and metastasis. Without blood vessels the maximum volume that can be attained by a tumor is ~1mm³; recruitment of new blood vessels through angiogenesis and/or vasculogenesis is therefore essential for tumor growth. It is now evident that these new blood vessels are formed by activation and proliferation of vessel wall stem/progenitor cells (VWSPC) or mature endothelial cells, both derived from local blood vessels, rather than by cells originating in bone marrow (references).

Understanding the contribution of various cellular components to tumor establishment, growth, invasion, and metastasis will require development of tools that can specifically ablate each of the cell types in vivo or in an ex vivo organ culture model. ILY-mediated cell ablation can be applied to both situations, particularly to an ex vivo organ culture model of new blood vessel formation. In the ex vivo aortic ring organ culture model, for example, abdominal aorta from mice is cut into ~1mm thick circular pieces and embedded into collagen in endothelial cell growth media with or without serum growth factors such vascular endothelial growth factors (VEGF). The aortic ring contains all layers of a large blood vessel, including endothelium, intima, smooth muscle, and adventitia, which acts as a reservoir of diverse types of cells, including VWSPC cells and tissue resident macrophages. Collagen-embedded aortic rings can sprout cells and form capillary-like structures when stimulated with growth factors and/or in the presence of appropriately sized tumor organoids. Formation of new capillaries begins with cell sprouting from the aortic rings within the first 24–48 hours. These cells then organize into capillary-like structures that can branch into a capillary network. It is likely that not only VWSPC and/or endothelial cells but also cells in the intima and particularly in the adventia play important roles in capillary formation. Deployment of rapid, inducible, and cell-type specific cell ablation can facilitate understanding of the role that each of these cell types plays in the formation of new blood vessels. The speed of ablation is important because the first stage of new capillary formation, activation of VWSPC or endothelial cells, occurs within 24–48 hours of culture. Techniques that require prolonged incubation of the inducer will therefore not be useful. Diphtheria toxin-based cell ablation, for example, cannot be used because it requires nearly three days, when capillary formation in aortic ring organ culture model is already well established

Cell ablation has been used extensively to investigate processes related to development and regeneration in several model systems from worm to mice (Table 1). Before the advent of genetic cell ablation methods, laser ablation was used to examine the role of the anchor cell in vulval induction and uterine development in C. elegans ^{147, 148149}, and of floor plate cells and motor neurons in neural patterning in the zebrafish embryo ^150151152 Moreover, optogenetic cell ablation is the preferred method for transecting axons in axon regeneration studies in worms and zebrafish (Table 1 and Fig. 1) ^{153154155156157} Genetically-mediated cell ablation has been used extensively in zebrafish in several contexts: 1) to ablate putative stem cells and study their roles in the spinal cord and fins ¹⁵⁸¹⁵⁹; 2) to ablate endothelial cells and study their role in regenerating cardiac tissue ^{88, 160}; and 3) to ablate motor neurons and evaluate their contribution to specific behaviors ¹⁵ In Drosophila, genetically-mediated cell ablation has been employed to study neuronal circuits ¹¹²⁶ The various chemical- genetic ablation methods used in these studies require several hours to be fully effective and therefore are not suited for investigating dynamic events like cell motility and neuronal activity. Due to its rapid action, ILY-hCD59 mediated cell death represents a powerful alternative approach to generating fundamental insights into dynamic cellular processes and functions (Fig. 4).

Cell ablation tools are not normally used to create mouse models of human diseases because only a few are caused by deficiency of a single cell lineage. Examples include type I diabetes, which results from loss of pancreatic β-cells; Parkinson’s disease, from loss of dopaminergic neurons; hemolytic anemia, from loss of mature erythrocytes; ribosomopathies such Diamond-Blackfan anemia, from loss of myelogenic progenitors. Streptozotocin (STZ)-induced type I diabetes¹⁶¹ and chemically induced Parkinson’s disease ^{41, 42} have been extensively used to study the pathogenesis of these diseases. STZ specifically targets and damages pancreatic p-cells by interaction with the GLUT2 receptor¹⁶², which is abundant on p-cell plasma membranes, but also expressed in liver and kidney to a lesser extent¹⁶². Caspase-mediated cell ablation has generated an animal model of inducible β-cell destruction, which is potentially useful in many areas of diabetes research (Wang ZV et al., 2008). As discussed earlier, injection of MPTP, paraquat, 6-OHDA or rotenone to mammalian CNS eliminates dopaminergic neurons and provides animal models of Parkinson’s disease^{41, 42}

Generation of a model of intravascular hemolysis in mouse is important for better understanding the pathogenesis of the sequelae of intravascular hemolysis in human hematological diseases. These sequelae include such important clinical manifestations as abdominal pain, dysphasia, erectile and endothelial dysfunction, pulmonary arterial hypertension (PAH), renal failure, platelet activation/thrombosis ¹⁶³, and even unexpected sudden death^164–167. This accounts for 12–26% of all deaths in sickle cell disease (SCD)^168–172 and occurs frequently in other acute and chronic hemolytic diseases, such as thalassemia, hemolytic uremic syndrome (HUS), paroxysmal nocturnal hemoglobinuria (PNH), ABO mismatch transfusion reaction, malaria, and autoimmune hemolytic anemia^173–177. Pulmonary arterial hypertension (PAH) is suspected to be a strong determinant of mortality in hemolytic disorders^178–180, although high mortality with only mild-to-moderate increases in systolic pulmonary artery pressure remains an unresolved paradox¹⁸¹. Acute hemolytic disorders such as ABO mismatch transfusion reaction and HUS associated with escherichia coli O157:H7 infection are less commonly seen in clinic than chronic hemolytic disorders, but also are associated with sudden death^174–177. The underlying mechanism by which hemolysis contributes to the development of PAH in SCD and other hemolytic disorders still remains unclear^{182, 183} Additionally, the risk factors for a fatal attack are poorly defined due to the lack of a rapid, conditional hemolysis model^{52, 53} To address these questions, we developed an unique hemolysis model, in which sudden death and varying degrees of hemolysis can be conditionally induced in the ThCD59^RBC by administration of ILY to mice⁹⁸ and rats ⁵⁴ We used this approach to demonstrate the critical roles of NO insufficiency and platelet activation in the pathogenesis of PAH and sudden death in the acute massive hemolytic model ¹²² These results also show that ILY-mediated rapid erythrocyte damage model is very useful for dissecting the pathogenesis of hemolysis-associated complications.

Most of the cell ablation techniques, except DT/DT-A, can be applied to study the function of targeted cell populations in large animal species such as monkey, pig, and rabbit. The DT/DT-A method cannot be used in animals other than mice and rats because other species express an endogenous DT receptor that makes their cells sensitive to DT-mediated cell apoptosis. The ILY-induced technique, however, can be used to generate cell ablation models in a large animal species ⁹⁸ Broad use of this strategy in species besides mice and rats is achievable due to the following specific features⁹⁸: (1) ILY does not damage cells in animals other than humans¹¹¹ and (2) Absence of ILY-neutralizing antibodies (Abs) in species other than humans allows it to be used in transgenic animals such as mice, rats, and pigs⁹⁸. Importantly, hCD59 overexpression caused no obvious abnormalities in hCD59 transgenic mice ^{98, 122}