Novel immunodeficient Pde6b rd1 mouse model of retinitis pigmentosa to investigate potential therapeutics and pathogenesis of retinal degeneration Alaknanda Mishra1, Barun Das1x, Madhu Nath2x, Srikanth Iyer1x, Ashwani Kesarwani1x, Jashdeep Bhattacharjee1, Shailendra Arindkar1, Preeti Sahay1, Kshama Jain1, Parul Sahu1, Prakriti Sinha1, Thirumurthy Velpandian2, Perumal Nagarajan1, Pramod Upadhyay1* National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067 Department of Ocular Pharmacology, Dr.Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi 110029 x: Authors who contributed equally * Correspondence: PramodUpadhyay, PhD National Institute of Immunology, ArunaAsaf Ali Marg, New Delhi 110067, India Email:pkumar@nii.ac.in, Phone : +91-11-26703770 ORCID iD: https://orcid.org/0000-0002-5560-822X Keywords:Retinitis pigmentosa, Mouse model, Immune compromised, Cell therapy Summary Statement NOD.SCID-rd1 is an immune compromised mouse model of retinitis pigmentosa (RP) to investigate cell based therapeutics for retinal rescue during RP and to study immunological © 2017 Published by The Company of Biologists Ltd This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed Biology Open • Advance article aspects of its pathogenesis and progression Abstract Retinitis pigmentosa (RP) is a common retinal degeneration disease caused by mutation in any gene of the photo transduction cascade and results in photoreceptor dystrophy Over decades, several animal models have been used to address the need for elucidation of effective therapeutics and factors regulating retinal degeneration to prohibit or renew the damaged retina However, controversies over immune privilege of retina during cell transplantation and role of immune modulation during RP still remain largely uninvestigated due to lack of proper animal models Therefore, in our present study, we have developed an immune compromised mouse model NOD.SCID- rd1 for retinitis pigmentosa (RP) by crossing CBA/J and NOD SCID mice and selecting homozygous double mutant animals for further breeding Characterization of the newly developed RP model indicates similar retinal degeneration pattern as CBA/J with decreased apoptosis rate and rhodopsin loss It also exhibits loss of T cells, B cells and NK cells NOD.SCID- rd1model is extremely useful for xenogenic cell based therapeutics as indicated by higher cell integration capacity post transplantation The dissection of underlying role of immune system in the progression of RP and effect of immune deficiency on immune privilege of eye has also been further elucidated Biology Open • Advance article using comparative qPCR studies of this model with immune competent RP model Introduction Retinal differentiation and maturation is a strictly regulated process in human beings(Yang 2004).The retinal degeneration diseases are irreversible once the retinal cells have degenerated because the adult retina is considered to lack stem cells and the cells lost are never regenerated(Jeon et al 1998) To address this need, the recently emerging field of regenerative medicine seems to be promising where different sources of pluripotent and somatic cells are reprogrammed into a specific cell type and transplanted into the site of defect (Bharti et al 2014a;Ouyang et al 2016;Siqueira 2011) Though these studies remain in the initial phase, it is expected that this may open newer therapeutic options for the retinal degeneration diseases Over many decades, animal models have been frequently used to elucidate the factors regulating the retinal degeneration and to develop ways to prohibit or renew the damaged retina Researchers have used a variety of retinal degeneration models for the same according to the purpose of their study(Chang et al 2002;Chang 2013;Veleri et al 2015) Pde6b rd1 mouse model is one of the successfully used and widely characterized mouse models for retinitis pigmentosa (Chang 2013;Veleri, Lazar, Chang, Sieving, Banin, & Swaroop 2015) It shows an early onset of retinal degeneration starting from weaning age due to a xenotropic murine leukemia viral insert (Xmv28) in the first intron of Pde6b and a non specific 3', 5’-cyclic phosphodiesterase subunit-β is an enzyme that is encoded by the Pde6b gene Since eye is also considered an immune privileged site, it has been a trend to use immune competent mouse models for cell based transplantation studies(Masli and Vega 2011;Taylor 2016).While the immune privilege stands true for some instances, mostly for the anterior Biology Open • Advance article specific mutation in 349th base pair of exon in Pde6b gene(Chang 2013) Rods cGMP- chamber of the eye, it is not an absolute phenomenon and its mechanisms still remain poorly dissected (Forrester and Xu 2012;Hori et al 2010;Taylor 2016).There is also the risk of immune cell penetration towards posterior chamber of eye as blood retinal barrier loses its integrity due to loss of photoreceptor and retinal pigment epithelial (RPE) cells that can lead to immune rejection or immune cell targeted loss of transplanted cells (Forrester & Xu 2012;Xian and Huang 2015a).The ability of adaptive and innate immune reactions to weaken engraftment of stem cell transplants is an important aspect of the host reaction that can affect the efficiency of cell transplantation(Cibelli et al 2013) Although a lot has already been chalked out about the pathogenesis of the disease (Berson et al 2002;Camacho and Wirkus 2013;Chang, Hawes, Hurd, Davisson, Nusinowitz, & Heckenlively 2002;Chang 2013;Veleri, Lazar, Chang, Sieving, Banin, & Swaroop 2015;Wright et al 2010), but only a little is known about the role of immune system in the progression of RP as it is mainly considered a hereditary disease Alterations in retinal homeostasis secondary to aging, metabolic abnormalities, altered vascular perfusion, or degenerative genetic conditions may initiate various inflammatory cascades that result from the breaching of posterior eye compartment due to breakdown of blood retinal barrier that sheaths the ocular environment from immune response (Forrester & Xu 2012;Hori, Vega, & Masli 2010;Whitcup et al 2013) in degeneration and inflammation Not much is known of the individual effect of adaptive or innate immunity in retinal degeneration and progression during RP The evaluation of such conditions may however become restricted due to unavailability of animal models that mimic the condition in which immune cells are absent so that a proper comparison of disease progression may be devised Biology Open • Advance article Moreover, it is of further importance to dissect out the part of immune system that is involved C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an Hence in our present study, we have developed an immune compromised mouse model of RP lacking in the function of Pde6b (functions in phototransduction cascade) and Prkdc gene (which encodes the catalytic subunit of the DNA-dependent protein kinase (DNA-PK) The homozygous Pde6b-/-Prkdc-/- mouse model was named as NOD.SCID-rd1 where NOD.SCID indicates lack of T, B and NKT cells and rd1 stands for Pde6b-/- retinal degeneration model The proposed model mimics the diseased condition (RP) making it more apt for analyzing the role of immune cells in retinal degeneration and the pros and cons of the cell based Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn Biology Open • Advance article therapeutics C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an RESULTS Hematology All the hematological parameters of NOD.SCID- rd1mice were comparable to CBA/J mice except total leukocytes and lymphocytes, which were significantly lower in NOD.SCID- rd1 as against BALB/c and CBA/J (Fig 1A).However, as compared to the NOD SCID mice, it showed no significant changes in leukocyte-lymphocyte proportion or any other parameters like hemoglobin, MCH and MCHC (Fig 1B) Genotyping Pde6b mutation screening Pde6b gene amplification resulted in a 485 bp product in all the wild type and Pde6b mutated animals (Fig 1C) The obtained product was further digested by HpyCH4IV restriction enzyme (cut site for HypCH4IV is A˅CG˄T) Since a non sense mutation caused in the 349th bp of exon in Pde6b gene(C to A) changes the sequence to AAGT, therefore the PCR product of the mutated animal does not get digested unlike wild type Hence, upon digestion, the wild type gene gives a 316bp and 169bp fragment while the heterozygote gives three fragments each of 485bp, 316bp and 169bp and mutated gene does not digest at all (Fig 1D) Prkdc mutation screening chain reaction with confronting two pair primers (PCR–CTPP) assay Here, mutation causes T to A single nucleotide transversion in exon 85 of the Prkdc gene This method of genotyping employs amplification of different regions of gene for mutated and wild type allele Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn Biology Open • Advance article Genotyping of SCID mice (Prkdc gene mutated) was done by allele specific polymerase C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an The SCID homozygous gene yields two fragment products at 257bp and 180 bp, wild type gene shows two fragments at 257bp and 101bp while heterozygotes show three bands at 257 bp, 180 bp and 101 bp owing to one mutated and one wild type allele (Fig 1E) Immune cell analysis in NOD.SCID- rd1 mice The levels of CD3, CD4, and CD8, B220 and NKT + cells were evaluated in peripheral blood and spleen of NOD.SCID- rd1 as compared to CBA/J and NOD SCID It was observed that there was no significant difference in the expression of either of these cells between NOD.SCID- rd1 and NOD SCID while CBA/J showed a significantly higher proportion of CD3, CD4, CD8 and B220 positive cells as compared to both NOD SCID and NOD.SCIDrd1 in peripheral blood Surprisingly, the NKT cell levels of CBA/J mice were greatly reduced in comparison to normal vision strains like BALB/c However, NOD SCID mice showed yet lower proportion of NKT + cells while it remained close for CBA/J and NOD.SCID- rd1(Fig 2A,B) The immune cell analysis in spleen revealed that CD3, CD4, CD8 and B220 positive cell population in NOD SCID mice was greatly ameliorated While CD3 positive cells showed no significant difference in NOD.SCID- rd1 as compared to CBA/J, the CD4 and CD8 positive cells were distinctly reduced in NOD.SCID- rd1indicating extreme alleviation of cytotoxic and helper T cells B220 and CD8 positive cells showed an upregulation in CBA/J which may retinal degeneration condition However, the NOD.SCID- rd1 mice displayed levels of B220 positive cells comparable to NOD SCID and significantly lower than CBA/J NKT cells remained without any significant change in both CBA/J and NOD.SCID- rd1 NOD.SCIDrd1 exhibited extremely higher levels of CD14 (marker for macrophages) and CD11c (marker for dendritic cells), the major innate immunity components, as compared to CBA/J The Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn Biology Open • Advance article point towards the role of self or auto antibody production during RP which further progresses C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an result also suggests that B cells and cytotoxic T cells (CD8+) have a greater role to play during retinal degeneration owing to their extremely increased expression in CBA/J(Fig 2C) Relative quantification of immunoglobulin secretion Immunoglobulin levels of NOD.SCID- rd1 (IgA, M, G1, G2a, G2b and G3) were quantified relative to NOD SCID and CBA/J mice The result suggested that there was no significant difference between immunoglobulin levels of NOD SCID and NOD.SCID- rd1 mice, however immunoglobulin levels of CBA/J was significantly higher than both the strains This indicates that B cells are mostly absent in NOD.SCID- rd1 mice same as NOD SCID, and are non functional even if a small percentage of B cells remained (Fig 2D) Qualitative analysis of spleen The spleen size of NOD SCID was the least amongst all the strains while BALB/c exhibited largest spleen size NOD.SCID- rd1 had an intermediate size to that of CBA/J and NOD SCID (Fig 2E) Quantitative RT-PCR analysis for retina specific genes Rod specific genes revealed significant alleviation in the m-RNA level in CBA/J as compared to BALB/c (normal vision phenotype) NOD.SCID-rd1 however shows an intermediate level of Rhodopsin and Recoverin expression demonstrating that NOD.SCID-rd1undergoes rod matrix of the retina between the retinal pigment epithelium and the photoreceptor cells does not show any change in expression suggesting that retinoid transport remains unaffected during RP Moreover, cone photoreceptor markers (S-opsin, Rom-1 and Peripherin) and bipolar cell markers (Pkc-α and Chx-10) show unaltered expression in both CBA/J and NOD.SCID- rd1 with respect to BALB/c suggesting that these retinal cell types not get Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn Biology Open • Advance article cell degeneration to a lesser extent than CBA/J Irbp, found in the interphotoreceptor C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an affected by rod cell degeneration at initial stages CBA/J and NOD.SCID-rd1 also exhibit higher m-RNA levels in amacrine cell markers (Cralbp, Dab-1)and retinal microglia markers (Gfap, S-100 and CD44)suggesting an over active cytokine/chemokine pathway and an increased gliotic index during RP Retinal pigment epithelium (RPE) marker (Rpe-65) remained unaffected while Mitf showed an elevated expression in both CBA/J and NOD.SCID-rd1 Retinal ganglion cell (RGC) marker (Brn-3a) was highly upregulated in NOD.SCID-rd1 while a slightly increased expression in CBA/J was also noticed This may suggest that ganglion cells which are the only output cells of the retina are well preserved and even upregulate during RP to compensate for the vision loss by enhanced output of signals to brain (Fig 3A) Western immunoblotting analysis The protein levels of the retina specific markers were checked by western blotting The results obtained convey that rod specific marker (RHODOPSIN) protein expression was absent while the expression levels of cone specific marker (S-OPSIN) was unaffected as compared to BALB/c at same age (4 weeks) in both CBA/J and NOD.ACID-rd1 The rod bipolar cell marker (PKC-α) showed attenuated expression in CBA/J confirming its simultaneous but slower degeneration with rod cells while NOD.SCID- rd1 exhibited normal expression of PKC-α PAX-6, a common retinal progenitor cell marker showed increased expression in CBA/J and attenuated expression in NOD.SCID- rd1 as against BALB/c cells respectively, were highly upregulated suggesting that they might be involved in the exacerbation of retinal degeneration during RP (Fig 3B, C) Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn Biology Open • Advance article Nevertheless, CRALBP and GFAP, which are markers of amacrine cells and muller glial C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an Immunofluorence analysis of photoreceptor markers Analysis for rod cell markers (Rhodopsin) and cone cell markers (S-opsin) was done to compare the level of degeneration of photoreceptor cells in CBA/J and NOD.SCID-rd1 It was observed that BALB/c (a normal vision control) exhibited proper outer segment staining for rods and cones while there was no staining observed for CBA/J and NOD.SCID-rd1 showed fewer cells expressing rod and cone markers in inner nuclear layer (INL) and ganglion cell layer (GCL) of retina Müller cells, which act as retinal progenitors during degeneration can be the reason behind the little expression of these photoreceptor markers in degenerated retina inNOD.SCID-rd1 (Fig 4A) Histopathological analysis of retinal degeneration during RP Histological analysis of the retina was performed by H and E staining BALB/c mice were used as a normal vision control The CBA/J and NOD.SCID- rd1 animals exhibited complete loss of outer segment (OS), photoreceptor (PR) layer and outer nuclear layer (ONL) at weeks of age However, inner nuclear layer (INL) and ganglion cell layer (GCL) remain intact This indicates that pde6b mutation affects not only rod photoreceptor cells but also cell types in close contact to them viz cone photoreceptor cells, bipolar cells or horizontal cells At the age of weeks, the density of the remaining INL still lowers, suggesting a time NOD.SCID- rd1(Fig 4B) Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn Biology Open • Advance article dependent progression of retinal degeneration and cell apoptosis in both CBA/J and C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an Figure : Fundoscopic imaging, ERG analysis of retina and apoptosis during RP (A, B, C): Representative fundoscopic retinal images for the analysis of retinal degeneration and vessel attenuation in BALB/c, CBA/J and NOD.SCID- rd1 respectively at weeks of age Both CBA/J and NOD.SCID- rd1 showed patched retina, however the severity of attenuated vessels and patchy appearance was aggravated in CBA/J as compared to NOD.SCID- rd1 BALB/c, which had normal vision, exhibited no patches and clear red vessels were visible throughout retina.(D, E): Electroretinogram analysis of BALB/c, CBA/J and NOD.SCID- rd1 at weeks of age BALB/c exhibited distinct a and b wave peaks while NOD.SCID- rd1 and CBA/J had no a and b wave peaks with reduced a wave latency indicating the degeneration of photoreceptors (rods and cones) as well as improper functionality of retinal bipolar cells.(F, G): Representative images illustrating apoptosis during retinal degeneration in CBA/J and NOD.SCID- rd1 as compared to normal vision BALB/c Ten random fields were scored for apoptotic cells in each case and graphically plotted The result referred to extremely high apoptotic rate of retinal cell in CBA/J While NOD.SCID- rd1 also shows apoptosis, it still remained significantly lower than CBA/J The green color stained cells represent the TUNEL positive Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn Biology Open • Advance article alexa fluor 488 stained apoptotic cells Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn Biology Open • Advance article C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an Figure : Behavioral analysis of NOD.SCID-rd1 model of RP (A, B): Behavioral analysis (n=10) by visual cliff test in bright as well as dim light showing that BALB/c mice preferred the shallow side of the cliff while both CBA/J and NOD.SCID- rd1 exhibited a 50-50% tendency to step on either side of the cliff (C, D) Light/dark latency test analysis indicated non aversive behavior of CBA/J and NOD.SCID-rd1 towards light that tended to spend more time in light chamber where they were initially placed However, BALB/c mice were aversive to light and tend to spend more time in dark chamber (E, F) The exploratory behavior of CBA/J and NOD.SCID- rd1 as indicated by the number of transitions made between both chambers as well as the delay in time taken to enter dark chamber from light chamber, is also reduced as compared to BALB/c (G) The spatial frequency was calculated based on optokinetic response of mice to various frequencies of gyrating stripes wherein CBA/J and NOD.SCID- rd1 failed to show any response [**** indicates p < Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn Biology Open • Advance article 0.0001, ** indicates p< 0.01 and * indicates p < 0.05] Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn Biology Open • Advance article C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an Figure 7: Effect of immune deficiency on immune privilege and transplantation (A): Macrophage marker MAC-1 displayed significant up regulation in CBA/J as compared to NOD.SCID- rd1 mice Neutrophil marker (Ly6G) was slightly high in CBA/J and natural killer (NK and NKT) cells were comparable in both CBA/J and NOD.SCID-rd1 (B): Pigment epithelial growth factor (PEDF) secreted by RPE cells was considerably elevated in NOD.SCID- rd1 as against CBA/J There was no significant change in the expression of vascular endothelial growth factor (VEGF) between CBA/J and NOD.SCID-rd1 (C): Matrix metalloproteases (MMP-1 and MMP-2) and monocyte chemoattractant protein (MCP-1) were upregulated in both CBA/J and NOD.SCID-rd1 as compared to control, however showed no significant difference between the two groups (D): The proinflammatory interleukins (IL-17, IL-7, IL-1b, IL-6, and IL-33) showed increase in both CBA/J and NOD.SCID- rd1 mice while there was no observed change in IL-10 expression in NOD.SCID-rd1 compared to control CBA/J exhibited significantly high expression of IL-17 as against NOD.SCID-rd1 (E): Another set of pro-inflammatory cytokines (TNF-a, IFN-g and TGF-b) also indicated upregulation in both RD models; however, IFN-g expression was considerably high in CBA/J (F): GFP +ve mouse retinal cells (1X10^6) were transplanted subretinally in the eye of groups of mice (4 Weeks): CBA/J- Wild type (CBA/J-WT), CBA/J-Immune suppressed (CBA/J-IS) and NOD.SCIDrd1 The representative confocal images (63X) of retinal sections are shown here GFP +ve cells integrated into the retina of all the three groups; however CBA/J-WT exhibited least engraftment with a few cells integrating into GCL and still fewer cells in INL CBA/J-IS mice showed significant cell integration into GCL and a few cells in INL A single line of ONL, seen above yellow dotted line, was also preserved and stained GFP +ve suggesting that donor photoreceptor cells survived and integrated into host CBA/J-IS retina NOD.SCID-rd1 exhibited maximum cell integration amongst the three groups of animals GFP +ve cells integrated in high numbers in GCL and a few in INL However, like CBA/J-IS, it also showed donor GFP +ve photoreceptor cell integration just above INL in (G): Graphical representation of cell engraftment quantification in the major layers of retina (ONL, INL and GCL) in each group of animals and their comparison to characterize potential cell based therapeutics of each group [**** indicates p < 0.0001, ** indicates p< 0.01 and * indicates p < 0.05] Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn Biology Open • Advance article significant numbers C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an Biology Open (2017): doi:10.1242/bio.021618: Supplementary information MATERIALS AND METHODS Animal Housing and breeding NOD.CB17-Prkdcscid/J (NOD SCID), CBA/J, BALB/cByJ (BALB/c) and transgenic C57/B6GFP (C57BL/6-Tg (UBC-GFP) 30Scha/J) mice were obtained from the Jackson Laboratory, USA The animals were housed in individually ventilated cages (IVC) with ad libitum access to acidified autoclaved water The animal room was maintained at 21-23 °C on a 14-h light-10 h dark cycle, and all procedures were carried out in accordance with the guidelines for care and use of animals in scientific research (Indian National Science Academy, New Delhi, India) in a committee for the purpose of control and supervision of experiment on animals (CPCSEA) registered animal facility 6-8 week-old female CBA/J mice having Pde6b mutation were crossed with 6-8 week old male NOD SCID mice The progeny of F1 generation were intercrossed, and all the F2 generation pups were screened for Pde6b and Prkdc mutation The homozygous double mutant phenotype was selected for further breeding program for over 15 generations to make a homozygous double mutant colony Tail biopsies (around mm) were obtained from 2-3 week old mice and were incubated overnight at 55oC in 600ul TNES buffer supplemented with 35ul proteinase K (10mg/ml) 166.7 ul of 6M sodium chloride (NaCl) was further added and shaken vigorously for 20 seconds The samples were then centrifuged at 12000 X g for minutes at room temperature (RT) The supernatant was collected and the DNA was precipitated with volume of chilled 95% ethyl alcoholfollowed by two washes with 70 % ethyl alcohol The DNA thus obtained was dissolved and resuspended in 100ul Tris-EDTA (TE) buffer Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn Biology Open • Supplementary information Isolation of crude DNA by high salt method C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an Biology Open (2017): doi:10.1242/bio.021618: Supplementary information Genotyping for screening of homozygous double mutant phenotype 1) SNP-RFLP (Single nucleotide polymorphism-restriction fragment length polymorphism) method for Pde6b mutation screening The oligonucleotide primers were prepared using Beacon Designer for the amplification of Pde6b gene present in exon 7, codon 349 of chromosome in mice which undergoes a single nucleotide polymorphism from cytosine (C) to adenosine (A) in the mutant phenotype PCR amplification was performed to yield a 485 bp product in all the wild type, heterozygous or homozygous mutant animals (Initial denaturation: 95 ºC for minutes, Denaturation: 95 degree for 30 seconds, Annealing: 52οC, 45 seconds for 30 cycles, Extension: 72 ºC for 30 seconds, Final extension: minutes) The amplicons were then digested with HypCH4IV whose cutting site is A˅CG˄T as per manufacturer’s protocol (NEB, UK) The digested product was run on 1% agarose gel and visualized in BioRad UV gel doc system Since the sequence becomes AAGT on mutation, the restriction enzyme failed to digest the amplified product in Pde6b mutated animal, however the digested PCR product yielded two bands in wild type (316 bp, 169 bp) and three bands in heterozygous animals ( 485bp, 316bb, 169bp) owing to only one defective pde6b allele for Prkdc mutation screening The screening of Prkdc gene involves two primer pairs carrying out simultaneous amplification of three different sizes of DNA segments in a single tube reaction Genomic DNA was subjected to PCR in a total volume of 20 ul containing 5X Firepolmastermix (Solis Biodyne) 1uM each of primers, DNA template and MilliQ Thermal cycling conditions were as follows: Initial Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn Biology Open • Supplementary information 2) PCR-CTPP (Polymerase Chain Reaction-Confronting Two Primer Pairs)method C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an Biology Open (2017): doi:10.1242/bio.021618: Supplementary information denaturation: 94 ºC, minutes; Denaturation: 94 ºC, 30 sec; Annealing: 60 ºC, 30 sec; Extension: 72 ºC, 20 sec; Final extension: 72 ºC, min; Cycles: 40 Immune cell analysis in NOD.SCID- rd1 mice 50 ul of anticoagulated whole blood was incubated with the respective antibody (1:1) at a dilution of 1:200 for 40 at room temperature (RT) After incubation, the red blood cell (RBC) lysis was performed and the cells were fixed with RBC lysis and fixing solution (BD Biosciences, USA) The spleen was crushed between two frosted slides and sieved using 100um filter (BD Biosciences, USA) to obtain a single cell suspension The antibodies used were FITCconjugated anti-mouse CD3, allophycocyanin (APC)-conjugated anti-mouse CD4, APC Cy7conjugated anti-mouse CD8, Phycoerethrin Cy5.5 (PE Cy 5.5) anti mouse B220 and Phycoerethrin (PE) conjugated anti mouse NKT (All from BD Biosciences, USA).Staining procedures were similar to that followed for peripheral blood Thereafter, samples were run on BD FACSVerse and analyzed in FACSDiva The analysis was performed in lymphocyte gated population Appropriate amount of each isotype specific rat anti-mouse purified monoclonal antibody (1:500) was coated overnight and the plate was further blocked with 5% BSA in PBS for hour at RT After washes with PBST for minutes each, samples at a dilution of 1:50 were added to the plate and incubated for hours at RT The plate was then washed thrice for minutes each with PBST Thereafter, detection antibody at a dilution of 1: 1000 was added to the plate and incubated for hour at RT followed by three washes with PBST (5 minutes each) In the next step, streptavidin HRP was added at a dilution of 1:4000 and incubated for 30 minutes at RT Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn Biology Open • Supplementary information Relative quantification of immunoglobulin secretion C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an Biology Open (2017): doi:10.1242/bio.021618: Supplementary information followed by washes for minutes each Finally, 100ul TMB substrate was added per well and allowed to incubate till color developed and the reaction was stopped by adding stop solution.Absorption maxima were measured spectrophotometrically at 450 nm and 570 nm Wavelength correction was performed by subtracting the readings obtained at 570 nm from 450 nm The readings obtained were plotted to obtain a comparative result for total immunoglobulin secretion Quantitative RT-PCR analysis for retina specific genes and immune privilege markers Total RNA was extracted from the whole eye tissue of mice by TRI Reagent (Sigma, USA) cDNA synthesis was performed by Verso cDNA Synthesis Kit (Thermo Fisher Scientific, USA) using 1µg total RNA and quantitative real-time PCR was performed usingDyNAmo Flash SYBRGreen (Thermo Fisher Scientific, USA) on the Master cycler Realplex platform (Eppendorf, Germany) The PCR conditions used were as follows: initial denaturation 95ºC for minutes, followed by 40 cycles of 95ºC for 15 seconds (denaturation), 60ºC for minute (annealing and extension) Table 1: List of primers Forward primer Reverse primer Pde6b 5’ GGTATCATGAATGAAGAA 3’ 5’ CACTAACCTACTTAACCT 3’ Rhodopsin 5’ GTCCACTTCACCATTCCTA 3’ 5’ TTCCTTCTCTGCCTTCTG 3’ Recoverin 5’ TCGTCATGGCTATCTTCAA 3’ 5’ TGGATCAGTCGCAGAATT 3’ IRBP 5’ GCTGCTGGTAGAACACAT 3’ 5’ CACAAGGCTGAGATGGAG 3’ S-opsin 5’ TACTTGGATTATTGGTATCG 3’ 5’ ACAGAAGATGAAGAGGAA 3’ Rom-1 5’ ATGGCTACAAGGATTGGT 3’ 5’ GGATTACAACAGGAGAAGG 3’ Peripherin 5’ GAGAAGGTGGTGACAGAG 3’ 5’ GCTTAGGAATAGGCTGAGA 3’ Gfap 5’ CAAGGAGATACTCTGAAC 3’ 5’ GTGAAGCAATAGAACTCT 3’ S100 5’ GCTGTGGACAAGGTAATGAAGGAA 3’ 5’ AGCCACCAGCACAACATACTC 3’ Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn Biology Open • Supplementary information Gene C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an CD44 5’ ACAACTTCTGGTCCTATGA 3’ 5’ CCACACCTTCTCCTACTAT 3’ IL-17 5’ CTCACACGAGGCACAAGT 3’ 5’ GCAGCAACAGCATCAGAGA 3’ IL-7 5’ ATCCTTGTTCTGCTGCCTGTC 3’ 5’ TTCGGGCAATTACTATCAGTTCCT IL-10 5’ CTGGGTGAGGAAGCTGAAG 3’ 5’ CCACTGCCTTGCTCTTAT 3’ IL-1b 5’ AAGGGCTGCTTCCAAACC 3’ 5’ GATGTGCTGCTGCGAGAT 3’ IL-6 5’ CGCTATGAAGTTCCTCTC 3’ 5’ TCTGTGAAGTCTCCTCTC 3’ IL-33 5’ TGCATGAGACTCCGTTCTGG 3’ 5’ TCCCGTGGATAGGCAGAGAA 3’ Mac-1 5’ TTCACGGCTTCAGAGATG 3’ 5’ CCATACGGTCACATTGTTG 3’ Ly6G 5’ CGTTGCTCTGGAGATAGAAGTTA 3’ 5’ GTTGACAGCATTACCAGTGAT 3’ CD14 5’ GAAGCCAGAGAACACCAC 3’ 5’ CAACAGCAACAAGCCAAG 3’ NK 5’ GGTTCTGGACAAGATGAAGT 3’ 5’ CGATGCCGATGTTGATGA 3’ NKT 5’ CTCACGCTCAAGGCAATCA 3’ 5’ CCGAGTCCAGACCTCCATT 3’ PEDF 5’ CTTACGATACGGCTTGGA 3’ 5’ CTTGGATAGTCTTCAGTTCTC 3’ VEGF 5’ CGACAGAAGGAGAGCAGAAG 5’ CTCAATCGGACGGCAGTAG 3’ MMP-1 5’ TATTGTTGCTGCCCATGA 3’ 5’ TTGCCAGTGTAGGTATAGATG 3’ MMP-2 5’ GACCACAACCAACTACGATGA 3’ 5’ GCTGCCACGAGGAATAGG 3’ MCP-1 5’ CTACTCATTCACCAGCAAGAT 3’ 5’ TCAGCACAGACCTCTCTC 3’ TNF-a 5’ CACCACCATCAAGGACTCA 3’ 5’ GGCAACCTGACCACTCTC 3’ IFN-g 5’ CTGAGACAATGAACGCTACAC 3’ 5’ TCTTCCACATCTATGCCACTT 3’ TGF-b 5’ AGCTTTGCAGGGTGGGTATC 3’ 5’ GGGGCCATTCACTAGCAGTT 3’ Western immunoblotting analysis After euthanasia by cervical dislocation, eyes were enucleated from mice and homogenized at 10 %( w/v) in ice cold RIPA buffer supplemented with a protease inhibitor cocktail (PIC) Protein content of the homogenate was estimated using bicinconinic acid (BCA) kit (Pierce,Rockford,IL) 50µg of protein was denatured and resolved on 12% SDS gel and transferred on a PVDF membrane Immunochemical staining was performed using primary antibodies at a dilution of 1:1000 and the membrane was incubated overnight at ºC Further Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn Biology Open • Supplementary information Biology Open (2017): doi:10.1242/bio.021618: Supplementary information C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an Biology Open (2017): doi:10.1242/bio.021618: Supplementary information incubation of the membrane with secondary antibodies (dilution 1: 5,000) was performed for hours at RT followed by washes for a total of 30 minutes with PBST Bands were visualized with the enhanced chemiluminiscence kit according to manufacturer’s protocol (Biorad, USA) Immunocytochemistry for the analysis of photoreceptor cells Whole eyewas enucleated after euthanizing the animals by cervical dislocation The tissue was fixed overnight in 4% paraformaldehyde (4% PFA), processed in 30% sucrose and embedded in OCT (optimal cutting temperature) medium Tissue sections of micron thickness were obtained on poly-l-lysine (Sigma Aldrich, USA) coated slides using cryotomeand stained with rod and cone retinal cell specific markers Rhodopsin and S-opsin (Thermo fisher scientific, USA) at a dilution of 1:100 for hour at RT Secondary antibody was added for 40 minutes at a dilution of 1:200 followed by washes with PBST, minutes each The slides were again washed thrice and nucleus staining was done using DAPI for minutes at a dilution of 1:500 The representative confocal images were taken at 63 X magnification using a system incorporated in the microscope (Zeiss LSM Version 4.2.0.121) Fundoscopic retinal imaging The animals were dark adapted for 40 minutes before the imaging procedure Thereafter, they the body weight) Upon dilation, the mice were laterally placed on the stand with their eye focused towards the camera The retina was focused with the objective lens and posterior pole images of both eyes were captured using streampix software using MICRON III rodent imaging system (Phoenix Research Labs, USA) Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn Biology Open • Supplementary information pupils were dilated using 1% tropicamide and anaesthetized using ketamine-xylazine (80mg/kg C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an Biology Open (2017): doi:10.1242/bio.021618: Supplementary information Electroretinography (ERG) Mice were dark-adapted for at least 12 hoursand anaesthetized After pupil dilation (1 drop Atropine, 1%), individual mice were fixed on a movable sled and gold wires(as active electrodes) were placed on the cornea Theground electrode was inserted in the tail and a reference electrode was placed subcutaneously between theeyes near cornea The retina was stimulated using white light of 1cds/m2and the mean of 25 averages was taken for obtaining single ERG response from the mouse retina The ‘a’ and ‘b’ wave amplitude and latency were measured in all groups, using inbuilt algorithm of labscribe software.The ERG responses were obtained through ERG attachment of MICRON III rodent imaging system using labscribe software (Phoenix laboratory, USA) Terminal deoxynucleotidyltransferasedUTP nick end labeling (TUNEL) assay for the analysis of apoptosis in RP Paraffin sections of whole eye tissue were obtained, deparaffinized and rehydrated by successive serial washings with ethanol and treated with proteinase K for permeabilization of cells followed by equilibration for 10 minutes at RT Fragmented DNA was labeled with terminal hour at 37°C in a humidified chamber.The reaction was stopped by immersing the slides in 2X SSC solution for 15 minutes and was counterstained by DAPI to visualize nuclei The image acquisition was performed under confocal microscope at 63X (Zeiss LSM Version 4.2.0.121) Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn Biology Open • Supplementary information deoxynucleotidyltransferase (TdT) and biotin deoxynucleotides (dNTPs) and incubated for C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an Biology Open (2017): doi:10.1242/bio.021618: Supplementary information Behavioral analysis Visual cliff test The visual cliff apparatus was prepared in a wooden box (62 x 62 x62 cm) with the four edges emerging 19 cm above the top and a wooden platform running along the middle The platform was 3.75 cm in height, 60 cm in length and 2.5 cm in width The platform separated the box into two sections, one with checker paper placed on the top surface of the box, whereas the other had the same checker paper placed on the bottom surface of the box The experiment was conducted by placing the mouse on the platform and observing the decision of mouse to step towards either safe zone or cliff zone in minutes Safe zone (shallow) results represented the events of the mouse stepped down to the reasonable side, which has the checker paper on the top surface of the box Cliff zone (deep) represented the events of the mouse stepped down on the “fake cliff” side, which has the checker paper on the bottom surface of the box 10 mice of each strain (BALB/c, CBA/J, and NOD.SCID- rd1) were used in this study and each mouse was tested for trials To reduce the effect of learning and memory, the apparatus was turned 180 degrees after two trials so that the “safe zone” and the “cliff zone” were at different sides for the next three trials The glass surface was cleaned thoroughly between each trial in order to prevent mice from safe/cliff) was calculated Light / dark latency test Light / dark latency test apparatus consisted of a box sized 21 X 42 X 25cm divided in two equal halves by a partition with a cm connecting door One chamber was brightly illuminated (approx 400 lux) and the other was kept completely dark with black background The animal was introduced in the light chamber (LC) and observed for five minutes Time spent by the animal in each chamber was recorded The number of transitions from light to dark chamber (DC) and vice Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn Biology Open • Supplementary information finding visual clues about depth The percentage of stepping towards each zone (shallow /deep or C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an Biology Open (2017): doi:10.1242/bio.021618: Supplementary information versa was also counted along with the initial time taken by the animal to enter the DC from LC where it was introduced Optokinetic response (OKR) The optokinetic drum with stripes of varying grating frequencies (0.03, 0.13, 0.26, 0.52 and 1.25 cpd) contains a wooden platform 15 cm above the base and 10 cm farther to the periphery of the drum (diameter: 63 cm; height: 35 cm) Movement behavior and head tracking were analyzed in ambient room light (illuminance = 400 lux; measured in the center of the cylinder with a digital luxmeter (MASTECH®) with a rotation speed of rpm and a spatial frequency of 0.1 cyc/deg Prior to the analysis, the mice were allowed to adapt to the environment of the non-rotating drum for ten minutes Thereafter the drum was rotated clockwise and anticlockwise, minute each for every stripe used with a time interval of 30 seconds between two rotations Occurrence of headtracking reflexes was judged by the observer during each minute period Head tracking was defined as horizontal head movement at the same rate and the same direction as the drum for at least 15° (as described previously) If the spatial frequency of the black and white stripes was increased, a threshold was reached beyond which no tracking movements of the head were than or equal to this threshold but below the next spatial frequency tested Cell transplantation in the retina and post transplantation engraftment analysis Retinal cells from GFP transgenic mice were isolated by digesting retina with 0.05% trypsin at 37○ C for 30 minutes with continuous agitation These cells were passed through 70 micron sieve to eliminate debris and centrifuged at 200g, and resuspended in DMEM/F-12 to obtain single cell suspension 1X10^6 cells were transplanted sub retinally into the eye of wild type CBA/J, immune suppressed CBA/J and NOD.SCID- rd1 mice (n=4) Thereafter, the animals were Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn Biology Open • Supplementary information detected The visual spatial resolution (visual acuity) of the animal was estimated to be greater C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn