Humán alkalikus foszfatázt termelő transzgenetikus nyulak előállítása lentivirus géntranszferrel = Creating human alkaline phosphatase producing transgenic rabbits by lentiviral gene transfer

SIV lentivírus vektorral GFP-t expresszáló transzgenikus alapító nyulakat állítottunk elő. Legjobb tudásunk szerint lentivírussal elsőként sikerült transzgenikus nyulat előállítanunk világszerte. Mivel azonban az alapító nyulak különböző mértékben mozaikosak, élő zöld utódnyúl nem született. Emiatt az előre nem látható fajspecifikus különbség miatt, a pályázatban eredetileg tervezett emlőszövetspecifikus promóter - humán szöveti nemspecifikus foszfatáz transzgenikus nyulak előállítására lentivírus transzgenezissel nem került sor. Az előmag mikroinjektálással korábban előállított transzgenikus nyulak tejéből, három különböző módon próbáltunk tisztított rekombináns humán szöveti nemspecifikus foszfatáz fehérjét izolálni. A tisztított rekombináns fehérje biológiai hatásának igazolása szepszis egérmodellen folyamatban van. | GFP expressing transgenic rabbits were created with SIV lentiviral vector. To our knowledge those are the first lentiviral transgenic rabbits worldwide. Since the founder rabbits were expressing the transgene in mosaic pattern to different extent, alive green rabbit did not born from any of the founders. Due to this species specific difference in the efficiency of lentiviral transgenesis, we did not try to establish lentiviral transgenic rabbits with the planned mammary gland specific promoter - human tissue non-specific alkaline phosphatase lentiviral construct. From the milk of the human tissue non-specific alkaline phosphatase expressing transgenic rabbits, which were created by pronuclear microinjection we tried to isolate the recombinant protein on three different ways. Evaluation of the biological activity of the purified recombinant human tissue non-specific alkaline phosphatase is in progress

The potential impact of new generation transgenic methods on creating rabbit models of cardiac diseases

Since the creation of the first transgenic rabbit thirty years ago, pronuclear microinjection remained the single applied method and resulted in numerous important rabbit models of human diseases, including cardiac deficiencies, albeit with low efficiency. For additive transgenesis a novel transposon mediated method, e.g., the Sleeping Beauty transgenesis, increased the efficiency, and its application to create cardiac disease models is expected in the near future. The targeted genome engineering nuclease family, e.g., the zink finger nuclease (ZFN), the transcription activator-like effector nuclease (TALEN) and the newest, clustered regularly interspaced short palindromic repeats (CRISPR) with the CRISPR associated effector protein (CAS), revolutionized the non-mouse transgenesis. The latest gene-targeting technology, the CRISPR/CAS system, was proven to be efficient in rabbit to create multi-gene knockout models. In the future, the number of tailor-made rabbit models produced with one of the above mentioned methods is expected to exponentially increase and to provide adequate models of heart diseases

Characterisation of eGFP-transgenic BALB/c mouse strain established by lentiviral transgenesis

Lentiviral technology is a powerful tool for the creation of stable transgenic animals. However, uncertainties have remained whether constitutive promoters resist long-term silencing. We used concentrated HIV-1 based lentiviral vectors to create stable transgenic BALB/c mice by perivitelline injection. In our vectors eGFP expression was driven by the human EF1alpha promoter. The established transgenic animals were analyzed for eGFP expression by in vivo fluorescence imaging, PCR, histology and flow-cytometry. eGFP expression showed even distribution without mosaicism; however, tissue-dependent differences of eGFP expression were observed. Up to the sixth generation only one newborn showed eGFP inactivation. eGFP + transgenic bone marrow cells efficiently provided long-term haemopoietic repopulation in radiation chimeras, regenerating all bone marrow-derived lineages with eGFP + cells with distinct eGFP expression profiles. The established eGFP + BALB/c mouse strain is expected to be extremely useful in various immunological experiments

Continental vs. tropical breed: Immunity comparison under heat stress conditions utilizing qRT-PCR technique

Understanding the responses of animals to seasonal heat stress on the genomic level has led to the identification of genes implicated in thermal stress reaction mechanisms. In this study, the relative gene expression of Interleukin-10 (IL-10), an anti-inflammatory cytokine and biomarker for heat stress-mediated immune modulation, was observed during the summer and winter seasons in continental and tropical sheep breeds, namely, Hungarian Indigenous Tsigai and White Dorper, employing quantitative real-time polymerase chain reaction (qRT-PCR). Temperature-humidity indices (THI) were calculated to assess heat stress levels. The results indicate that IL-10 CT-values were significantly higher during the summer, when heat stress prevails, in both sheep breeds compared to winter. While the White Dorper exhibited a higher numerical value for the summer relative gene expression ratio (16.2) compared to the Hungarian Indigenous Tsigai (12.3), no significant differences in CT values were observed between breeds or among sexes. These findings suggest the immune-adaptive characteristics of the two sheep breeds during seasonal heat stress. The variation of IL-10 gene expression levels between the two breeds can be attributed to their geographical origins; the White Dorper emerging from arid subtropical South Africa and the Hungarian Indigenous Tsigai Sheep thriving in the seasonally harsh Carpathian climate for centuries

The creation of a multiallele knockout genotype in rabbit using crispr/cas9 and its application in translational medicine

Nonrodent animal models have recently become more valuable in preclinical studies. The limitation of nonrodent animal models is that they must demonstrate relatively reliable and predictable responses in addition to representing complex etiologies of a genetically diverse patient population. In our study, we applied CRISPR/Cas9 technology to produce transgenic rabbits. This approach can be useful for creating genetically divergent and homogeneous populations for studies in translational medicine. NADPH oxidase 4 (NOX4) is a promising therapeutic target, as it is linked to several pathologies including stroke, atherosclerosis, and lung and kidney fibrosis. NOX4 knockout (KO) rabbit lines were created in order to study the in vivo effects resulting from a lack of NOX4 protein and loss of gene function. One of the knockout founders was a germline multiallelic knockout male. Its offspring segregated into three distinct NOX4 knockout and a wild-type lines. Mosaicism is a relatively frequent phenomenon in rabbit transgenesis. Our results point to the possible application of mosaicism in preclinical studies. However, careful planning and evaluation of results are necessary. The predicted off-target sites were studied as well, and no signs of off-target events were detected. © 2020 by the authors. Licensee MDPI, Basel, Switzerland

Baromfi-immunglobulinok lehetséges alkalmazásai a mikotoxin-környezetterhelések és a humánérintettség fókuszában

Az immunglobulinok a biológiai funkciójuk mellett önállóan is alkalmazhatók állat- és humándiagnosztikai, immunoassay alapú mérési eljárásokban, a profilaxisban és (immun)terápiákban is. A fenti célokra napjaink állatetikai szempontokból is előnyös „alternatívája” a madár-, tojássárgájából izolálható, poliklonális, ún. immunglobulin-Y. Fejlesztése, termelése költséghatékony, a komplexitás alacsony, és a termelt immunglobulin-Y az előnyös tulajdonságai miatt jól használható is immunoassay-ben vagy még inkább az orvosi terápiákban (elsősorban a passzív immunizálásban). Napjainkban már széles körben alkalmazzák (patogének vagy expresszált toxinjaik ellen, a bélrendszer megbetegedései, gyulladásai vagy metabolikus betegségek kezelésében stb.). Humándiagnosztikai felhasználása azonban még limitált, néhány marker mérése történik ilyen anyagok – mint valamilyen immunoassay-komponens – felhasználásával. Tanulmányunkban egy lehetséges, ma még kevésbé elterjedt alkalmazási területet mutatunk be. Napjainkban a környezetterhelés problémaköre egyre jelentősebb. Az emberi tevékenységek, az iparosodás következtében a környezeti változások a természetes környezetterhelők megjelenését – köztük az egyes penészgombák által termelt mikotoxin-behatásokat is – lokálisan és globálisan is fokozzák, ami (elsősorban a táplálkozás útján) már az emberi közösségeket is érinti. A behatások gyakran együttesen jelentkeznek, több mikotoxin hat egy időben az egyedre. A mikotoxinok – a bélcsatornán felszívódva és felgyűlve a szervekben, szövetekben – már elérhetnek olyan szinteket, amelyek akár élettani, akár viselkedésre gyakorolt hatásokat is kiválthatnak. Bár az expozíciós források (például gabona és feldolgozott élelmiszerek) vizsgálata már hatóságilag is szabályozott, az akkumuláció ténye, mértéke sok esetben nem vizsgált vagy nem vizsgálható, illetve gyakran nem kellően veszik figyelembe. A dedikált (antimikotoxin)-madár-immunglobulin-Y praktikusan a technika jellege miatt is alkalmazható lehetne mind a (deponálódott) mikotoxin(ok) kimutatására, mind immunterápiás (például mikotoxin-neutralizációs) célokra. Az endokrin diszruptor hatású mikotoxint a zearalenon példáján (szaporodásbiológiai és immunológiai hatások ismertetésével) demonstrálva, a zearalenonra specifikus madárimmunglobulin-fejlesztéseket és -lehetőségeket szeretnénk bemutatni, szorgalmazva ezzel a humánkimutatásban való alkalmazást, valamint olyan mérési rendszerek fejlesztését is sürgetve, amelyek alkalmasak lehetnek dedikált, akár a többszörös akkumuláció igazolására is. Orv Hetil. 2023; 164(39): 1527–1536

The potential impact of new generation transgenic methods on creating rabbit models of cardiac diseases

Since the creation of the first transgenic rabbit thirty years ago, pronuclear microinjection remained the single applied method and resulted in numerous important rabbit models of human diseases, including cardiac deficiencies, albeit with low efficiency. For additive transgenesis a novel transposon mediated method, e.g., the Sleeping Beauty transgenesis, increased the efficiency, and its application to create cardiac disease models is expected in the near future. The targeted genome engineering nuclease family, e.g., the zink finger nuclease (ZFN), the transcription activator-like effector nuclease (TALEN) and the newest, clustered regularly interspaced short palindromic repeats (CRISPR) with the CRISPR associated effector protein (CAS), revolutionized the non-mouse transgenesis. The latest gene-targeting technology, the CRISPR/CAS system, was proven to be efficient in rabbit to create multi-gene knockout models. In the future, the number of tailor-made rabbit models produced with one of the above mentioned methods is expected to exponentially increase and to provide adequate models of heart diseases

Effect of ad libitum feeding of Holstein Friesian calves on immunological parameters and molecular stress on a transcriptional level

Our aim was to compare the health and performance of ad libitum (ADLIB) and restrictedly fed Holstein Friesian heifer calves. Calves were selected to ADLIB (n = 13) and control (n = 13) groups randomly. The period of ADLIB feeding lasted for 3 weeks after colostrum supplementation. The calves in the control group received the same milk replacer, which was supplied according to the restrained feeding schedule of the farm. There was no difference between the two groups in weight, weight gain or movement activity, furthermore in the values of glucose, albumin, total protein, BHB, AST, oxidant and antioxidant status incl. dROM, PAT and OSI. The IL8 gene had higher levels (non significant, p > 0.05) of expression in the ADLIB group during the first 20 days of life, which indicates that ADLIB feeding might potentiate a stronger immune response to environmental stress. The IGF1 gene showed increased expression in the ADLIB fed group at almost all time points, however the difference was already detected on the first day of the study, indicating the importance of individual differences even within the same breed. During the first 10 days INS expression was higher in the restricted group, followed by a shift by day 20 and after, when the ADLIB group showed a higher relative expression level. The observed values describe a trend that, although not significant (p > 0.05), would seem to indicate that ADLIB feeding might potentiate a stronger immune response to environmental stress

Hungarian indigenous Tsigai, a promising breed for excellent heat tolerance and immunity

The adverse effects of climate change on sheep farming have become more noticeable in recent decades. Extensive efforts have been made to untangle the complex relationship between heat tolerance, animal health, and productivity, also to identify a resilient and economically suitable breed for selection that can be resilient to future climate change conditions. Using quantitative real-time polymerase chain reaction (qRT-PCR), we observed the seasonal variations in the expression of several important genes related to heat stress and immunity (HSP70, IL10, TLR2, TLR4, and TLR8) in three of the most widely kept sheep breeds in Hungary: The indigenous Tsigai, Hungarian Merino, and White Dorper. We found that the seasonal stressor affected the relative gene expression of all genes in this study. Notably, The Hungarian indigenous Tsigai was the most robust breed adapted to the Hungarian continental (hot summer, cold winter) environment, with excellent thermotolerance and immunity. Furthermore, despite suffering from heat stress in the summer, Hungarian Merino maintained their robust immune system well throughout the year