74 EFFECT OF SIZE AND CULTURE PERIOD OF FROZEN–THAWED BOVINE TROPHOBLASTIC VESICLES ON INTERFERON-τ SECRETION

2014 ◽  
Vol 26 (1) ◽  
pp. 151
Author(s):  
Y. Hashiyada ◽  
H. Takahashi ◽  
D. Yamaguchi ◽  
K. Imai ◽  
M. Geshi
Keyword(s):  
Significant Positive Correlation ◽  
Culture Media ◽  
Fetal Bovine Serum ◽  
Calf Serum ◽  
Culture Period ◽  
Fetal Bovine ◽  
Student’S T ◽  
Student’S T Test

Frozen–thawed bovine trophoblastic vesicles (bTV) derived in vivo could secrete interferon-τ (IFN-τ) at the same level as fresh bTV on Days 4 to 6 after thawing. However, amounts of IFN-τ decreased following continuous in vitro culture (Hashiyada et al. 2012 38th IETS). Co-transfer of frozen–thawed bTV improved pregnancy rate of embryos due to the effects of IFN-τ secreted by bTV (Hashiyada et al. 2008 41th SSR). However, the relation between bTV size and IFN-τ secretion level during culture has not been well documented. The objective of present study was to characterise the concentration of IFN-τ related bTV volume and culture period after thawing of cryopreserved bTV. The bTV were prepared from Day 16 elongating blastocysts recovered nonsurgically. The dissected trophoblastic fragment, 1 to 1.5 mm in width, was cultured using TCM-199 supplemented with 20% (vol/vol) fetal bovine serum and 0.1 mM β-mercaptoethanol. Formed vesicles after 24 h of culture were cryopreserved using D-PBS supplemented with 20% calf serum and 1.8 M ethylene glycol. After thawing, bTV were cultured individually with 100 μL/well/day until Day 2 (i.e. the day of thawing was defined as Day 0), and thereafter changed to 200 μL/well/day to termination at Day 10. Collection of culture media and measurement of bTV diameter were performed before cryopreservation and after thawing for every day. Interferon-τ in collected media was measured by radioimmunoassay. The estimated bTV volume was calculated based on the diameter. Data were analysed by Student's t-test. Nine fresh bTV before cryopreservation were used to assess the IFN-τ secretion for 24 h in relation to bTV volume. A significant positive correlation was observed between secreted IFN-τ (mean ± s.e.M, 19.9 ± 3.1 ng mL–1) and bTV volume (1.49 ± 0.6 mm3, r = 0.91; P < 0.01). Initial IFN-τ secretion from bTV cultured for 24 h after thawing was significantly decreased compared with that before cryopreservation (29.1 ± 2.1 ng mL–1 and 58.4 ± 4.8 ng mL–1; P < 0.01, n = 27). In continuous culture of bTV (n = 8), IFN-τ secretion increased gradually from Day 2 (23.1 ± 9.0 ng mL–1) to Day 4 (32.2 ± 8.4 ng mL–1), and then maintained this level until Day 7 (33.4 ± 14.9 ng mL–1). However, this amount of IFN-τ tended to decrease on Day 8 (24.8 ± 5.0 ng mL–1), 9 (16.5 ± 4.4 ng mL–1), and 10 (12.0 ± 1.7 ng mL–1). Interferon-τ secretion from bTV on Day 9 and 10 was lower than that on Day 3, 4, 5, 6, and 8, respectively (P < 0.05). Volume of bTV increased also from Day 2 (0.2 ± 0.1 mm3) to Day 5, 6 (0.8 ± 0.3 mm3) and 7 (0.7 ± 0.2 mm3). However, bTV volumes shrank drastically on Day 8 (0.3 ± 0.1 mm3), 9, and 10 (0.2 ± 0.1 mm3). In comparison with bTV during culture, volumes on Day 4, 5, and 7 were greater than those on Day 2 and 3, and volumes on Day 6 and 7 were greater than on Day 8, 9, and 10 (P < 0.05). These results indicate that the dynamics of IFN-τ secretion reflected the expansion or reduction of bTV in continued culture after thawing. Interferon-τ secretion might be related to bTV volume. Moreover, we reconfirmed that cryopreserved bTV highly express IFN-τ during 4 to 7 days after thawing.

78 INTERFERON-τ SECRETION OF CRYOPRESERVED BOVINE TROPHOBLASTIC VESICLES DERIVED FROM IN VIVO

2012 ◽  
Vol 24 (1) ◽  
pp. 151
Author(s):  
Y. Hashiyada ◽  
H. Takahashi ◽  
K. Imai ◽  
M. Geshi
Keyword(s):  
Continuous Culture ◽  
Culture Media ◽  
Calf Serum ◽  
Measure Concentration ◽  
Fetal Bovine ◽  
The Mean ◽  
Student’S T ◽  
Student’S T Test

The co-transfer of bovine trophoblastic vesicles (bTVs) prepared from in vivo recovered conceptuses is known to promote the successful implantation of embryos, which expected lower viability, through the effects of interferon-τ (IFN-τ) secreted by bTVs. We have reported that the pregnancy rate was improved for co-transferred embryos with frozen-thawed bTVs using the direct-transfer technique (Hashiyada et al. 2008, 41st SSR). However, the IFN-τ secretion level from cryopreserved bTVs is not well known. The objective of the present study was to measure concentration of IFN-τ released from frozen-thawed bTVs individually cultured in vitro. bTVs were prepared from elongating blastocysts 3 to 20 mm in length, following superstimulatory treatment and recovered on Day 16 post-AI, by dissection using a surgical blade. Each trophoblastic fragment, 1 to 1.5 mm in width, was cultured in a well of a 96-well plate using TCM-199 supplemented with 20% (v/v) fetal bovine serum and 0.1 mM β-mercaptoethanol. Formed vesicles after 24 or 48 h of culture were cryopreserved using D-PBS supplemented with 20% calf serum, 1.5 M ethylene glycol (EG) and 0.1 M sucrose or 1.8 M EG. After thawing, each bTVs was cultured for 2 days to compare IFN-τ secretion between the 2 cryoprotectants. Furthermore, transition of IFN-τ level was assessed in continuous culture until Day 10 (the day of thawing was defined as Day 0). The volume of culture medium was 100 μL well–1 day–1 until Day 2 and thereafter changed to 200 μL well–1 day–1 until termination. Exchange and collection of culture media were performed on Day 1, 2, 4, 6, 8 and 10. Collected culture media were stored at –30°C until use. IFN-τ was measured by RIA (Takahashi et al. 2005 Theriogenology 63, 1050–1060). Data were analysed by Student's t-test. Initial IFN-τ secretion from bTVs before cryopreservation did not differ between 24 and 48 h of culture period to form vesicles, 44.0 ± 2.9 (mean ± standard error of the mean, n = 64) and 52.8 ± 6.4 ng mL–1 (n = 27), respectively. IFN-τ secretion was no difference between the 1.5 M EG group and the 1.8 M EG group on Day 1 (41.2 ± 4.9 ng mL–1, n = 42 and 30.4 ± 2.2 ng mL–1, n = 31) and on Day 2 (38.0 ± 5.4 and 38.2 ± 4.5 ng mL–1), respectively. In the continuous culture group (n = 28), IFN-τ secretion tended to increase from Day 2 (25.2 ± 3.4 ng mL–1) to Day 4 (51.8 ± 12.3 ng mL–1) and 6 (55.4 ± 13.3 ng mL–1) (P < 0.05). However, this amount of IFN-τ on Day 6 significantly decreased on Day 8 (25.6 ± 2.7 ng mL–1; P < 0.05) and Day 10 (15.5 ± 2.2 ng mL–1; P < 0.01), gradually. These results indicate that cryopreserved bTVs could secrete IFN-τ at the same level as fresh bTVs on Day 4 to 6 after thawing and then these amounts of IFN-τ significantly decrease in vitro.

162 IMPROVED EMBRYO SURVIVAL AND QUALITY WITH EMCARE II

2006 ◽  
Vol 18 (2) ◽  
pp. 189
Author(s):  
A. Harvey ◽  
M. Lane ◽  
J. Thompson
Keyword(s):  
Calf Serum ◽  
Cell Number ◽  
Tunel Staining ◽  
Embryo Survival ◽  
Improved Outcomes ◽  
Student’S T ◽  
The Impact ◽  
Student’S T Test

Collection of embryos exposes them to a number of stresses, including light, air, and changes in temperature. Improvement of holding media to reduce the impact of handling stresses on the embryo during in vivo collection and transfer is therefore beneficial to ensure maintenance of viability following transfer. The aim of this study was to compare the effect of holding IVP-derived blastocysts at 25°C in Emcare I (ECMI, Emcare, Dallas, TX, USA) with those held in Emcare II (ECMII), a proprietry formulation designed to reduce in vitro-induced stress. In vitro-produced bovine embryos were generated using standard protocols. Blastocysts were randomly allocated to either ECMI or ECMII (ICPBio, Aukland, New Zealand) on Day 7 and were held at 25°C for a period of 24 h, after which they were cultured in Cook Bovine Blast (Cook Australia, Brisbane, Australia) supplemented with 10% fetal calf serum for 48 h. At 24 and 48 h, embryos were scored for hatching, and a cohort removed for TUNEL staining at each time point. Differences were analyzed by Student's t-test. At both 24- and 48-h culture, hatching rates tended to be higher for embryos held in ECMII than in ECMI (Table 1). The level of apoptosis at 48 h was reduced in blastocysts held in ECMII (P = 0.06). Moreover, the total cell number of hatched blastocysts at 48 h was significantly increased (1.5-fold) in those held in ECMII (P = 0.01). Results suggest that the formulation of ECMII improves the ability of IVP bovine blastocysts to re-expand and hatch following an imposed stress (25°C for 24 h). Furthermore, ECMII improves overall embryo quality through a reduction in the percentage of cells undergoing apoptosis as well as through increased cell numbers, evident 48 h following cessation of the stress. We suggest that Emcare II reduces the impact of (or increases the embryo's tolerance to and recovery from) an imposed stress, which, although severe in the present study, may provide improved outcomes following embryo transfer in field situations. Table 1. Hatching and apoptosis of blastocysts held at 25°C for 24 h in Emcare I or Emcare II This work was supported with funding by ICPBio (NZ).

scholarly journals Study on the growth promoting capacity of calf and fetal bovine serum for animal cells "in vitro" II: electrophoretic study and survey on the antiproteolytic activity of pools of calf and fetal bovine serum

1984 ◽  
Vol 26 (2) ◽  
pp. 97-104 ◽  
Author(s):  
Edda de Rizzo ◽  
Celidéia Aparecida Coppi Vaz ◽  
Inácio França Mendes ◽  
Anatércia Ferreira Bonfim Yano
Keyword(s):  
Bovine Serum ◽  
Culture Media ◽  
Fetal Bovine Serum ◽  
Calf Serum ◽  
Animal Origin ◽  
Electrophoretic Study ◽  
Fetal Bovine ◽  
Growth Promoting ◽  
Antiproteolytic Activity

Calf serum and fetal bovine serum present great variability as to its growth promoting efficiency (GPE). As supplement of culture media to cultivate cells of animal origin they stimulate the "in vitro" multiplication and maintain cell viability. When fourteen lots of calf sera of variable GPE had the total protein contents as well as the percentages of serum fractions determined, no significant differences that could possibly explain the variability of the GPE were observed. Evaluation of the antiproteolytic activity of nineteen lots of calf serum and eighteen serum lots of younger calves showed that the former exhibited lower antiproteolytic titers (1:40 to 1:80) than the latter (1:80 to 1:160). Twelve lots of fetal bovine serum studied in parallel, showed the highest concentration of antiproteolytic factors, with titers equal to 1:320. Sera of bovine origin, but not fetal sera, are usually heat-inactivated, what was demonstrated to be responsible for the decrease of the antiproteolytic activity of 75% of the lots tested. This could explain the inability of certain heat-inactivated sera in promoting multiplication of some cells "in vitro", as verified with primary monkey kidney cells. The results obtained in this study indicated the convenience of submiting each lot of serum to be introduced in cell culture to previous determination of its characteristics, such as growth promoting efficiency, antiproteolytic activity and also toxicity, absence of extraneous agents, etc., in order to minimize the possibility of using serum lots of questionable quality, thus preventing not only the loss of cell lines, but also undesirable and sometimes expensive delays.

scholarly journals Fetal Bovine Serum-Derived Extracellular Vesicles Persist within Vesicle-Depleted Culture Media

2018 ◽  
Vol 19 (11) ◽  
pp. 3538 ◽  
Author(s):  
Brandon Lehrich ◽  
Yaxuan Liang ◽  
Pooya Khosravi ◽  
Howard Federoff ◽  
Massimo Fiandaca
Keyword(s):  
Cell Growth ◽  
Extracellular Vesicles ◽  
Bovine Serum ◽  
Culture Media ◽  
Fetal Bovine Serum ◽  
Cellular Growth ◽  
Primary Astrocyte ◽  
Fetal Bovine ◽  
Cell Growth And Viability

It is known that culture media (CM) promotes cellular growth, adhesion, and protects explanted primary brain cells from in vitro stresses. The fetal bovine serum (FBS) supplement used in most CM, however, contains significant quantities of extracellular vesicles (EVs) that confound quantitative and qualitative analyses from the EVs produced by the cultured cells. We quantitatively tested the ability of common FBS EV-depletion protocols to remove exogenous EVs from FBS-supplemented CM and evaluated the influence such methods have on primary astrocyte culture growth and viability. We assessed two methodologies utilized for FBS EV removal prior to adding to CM: (1) an 18-h ultracentrifugation (UC); and (2) a commercial EV-depleted FBS (Exo-FBS™). Our analysis demonstrated that Exo-FBS™ CM provided the largest depletion (75%) of total FBS EVs, while still providing 6.92 × 109 ± 1.39 × 108 EVs/mL. In addition, both UC and Exo-FBS™ CM resulted in poor primary astrocyte cell growth and viability in culture. The two common FBS EV-depletion methods investigated, therefore, not only contaminate in vitro primary cell-derived EV analyses, but also provide a suboptimal environment for primary astrocyte cell growth and viability. It appears likely that future CM optimization, using a serum-free alternative, might be required to advance analyses of cell-specific EVs isolated in vitro.

Effect of culture conditions on artificial activation of porcine oocytes matured in vitro

1996 ◽  
Vol 8 (8) ◽  
pp. 1153 ◽  
Author(s):  
N Yamauchi ◽  
H Sasada ◽  
S Sugawara ◽  
T Nagai
Keyword(s):  
In Vitro Maturation ◽  
Fetal Calf Serum ◽  
Culture Media ◽  
Calf Serum ◽  
Treated Group ◽  
Culture Conditions ◽  
Culture Period ◽  
Subsequent Response ◽  
Artificial Activation

The effects of culture media used and culture period for in vitro maturation of porcine oocytes on their subsequent response to chemical and electrical activation, were investigated. Activated oocytes were identified by the presence of a pronucleus(ei) or cleavage. Porcine oocytes were cultured for 24, 30, 36, 42 and 48 h in TCM199 with Earle's salts (199) supplemented with 10% fetal calf serum (199-FCS) before electrical stimulation. Although few oocytes were activated after 24 h and 30 h of culture (5.4% and 6.1% respectively), the percentage of activated oocytes increased significantly to 93.2% after 42 h in culture (P < 0.05); however, when the culture period was extended to 48 h, there was a significant decrease to 56.7% (P < 0.05). Oocytes were also cultured in four types of media: (1) 199-FCS; (2) 199 supplemented with 5 mg mL-1 bovine serum albumin (199-BSA); (3) Kreb's-Ringer bicarbonate solution supplemented with 10% FCS (KRB-FCS); and (4) KRB supplemented with BSA (KRB-BSA). After 42 h of culture in each medium, the oocytes were electrically activated. Although rates of maturation of oocytes cultured in the four media were similar (74.0-80.8%), all oocytes except those cultured in 199-FCS failed to be activated. In addition, oocytes were cultured for 36, 42 and 48 h in 199-FCS and then stimulated by treatment with ethanol. Significantly fewer oocytes were activated in the chemically-treated group than in the electrically-treated group. These results indicate that culture conditions used for the culture of porcine oocytes in vitro are important with respect to their subsequent response to artificial activation.

179 EFFECT OF SERUM DURING CULTURE ON DAY 14 ELONGATED BOVINE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 191 ◽  
Author(s):  
J. Angulo ◽  
G. T. Gentry ◽  
R. A. Godke ◽  
K. R. Bondioli
Keyword(s):  
Gene Expression ◽  
Messenger Rna ◽  
Culture Media ◽  
Calf Serum ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Expression Levels ◽  
The Mean

It has been reported that the addition of serum to embryo culture media alters gene expression and triggers the development of large offspring syndrome. The objectives of this study were to determine gene expression levels in embryos cultured in the absence or presence of 5% calf serum and in vivo-derived (IVD) embryos and to determine the effects of serum on the length of elongated embryos. Abattoir-derived oocytes were obtained from a commercial provider and fertilized at 24 h of maturation with semen from a bull previously used for IVF. At 18 h post-insemination (hpi), embryos were denuded and groups of 15 presumptive zygotes were cultured in 30-μL drops of modified SOF medium with amino acids and 6 mg mL–1 of BSA (mSOFaa). At 72 hpi, cleavage rate was assessed and embryos were randomly allocated into 2 treatments: mSOFaa without and with 5% calf serum. Embryos were then cultured to 168 hpi and blastocyst rates were assessed and recorded. Blastocysts (n = 5 to 10) from each treatment were transferred into synchronized recipients, and Day 14 embryos were recovered 7 days post-transfer. Embryos were photographed, measured, and immediately stored at –80°C in a minimal volume of PBS + 0.1% polyvinyl alcohol. Messenger RNA was isolated using a Dynabeads mRNA Direct Kit™ (Invitrogen, Carlsbad, CA), and reverse transcription was performed using an iScript™ cDNA Synthesis Kit (Bio-Rad Laboratories, Inc., CA). Quantitative PCR was performed to determine the transcript abundance for COX6A, IFNT1a, PLAC8, IGF2R, and GAPDH for each sample. The GAPDH was used as a reference gene, and gene expression was calculated as a ratio of expression levels between each gene of interest and GAPDH. Expression levels for each gene were determined from standard curves generated by serial dilutions of PCR amplicons starting with 0.4 pg/reaction. Blastocyst development rates were higher in embryos cultured with serum compared with the nonserum treatment (14.9 and 7.4% respectively; chi-square, P < 0.001). Lengths of elongated embryos from the serum (3395.3 ± 414.7 μm) and nonserum (2784 ± 741.8 μm) culture treatments differed from the IVD (6297.7 ± 677.2 μm) treatment (mean ± SE; ANOVA, P < 0.0052). There were no differences in the mean expression levels for COX6A, IFNT1a, PLAC8, and IGF2R across treatment groups, but in the serum treatment, 3 out 11 overexpressed IFNT1a, 4 out of 11 overexpressed IGF2R, and 2 out of 11 overexpressed PLAC8, defined as being 2 standard deviations above the mean of the IVD treatment for each respective gene. In the in vitro-produced nonserum and IVD treatments, overexpression by this definition was not observed. Although mean expression levels were not affected by culture with serum under these conditions, very high expression of IFNT1a, IGF2R, and PLAC8 was observed in some embryos cultured with serum, but not in embryos cultured without serum or IVD embryos.

75 Improvement of Developmental Competence of Bovine In Vitro-Produced Embryos by Using Charcoal:Dextran-Stripped Fetal Bovine Serum on In Vitro Culture Media

2018 ◽  
Vol 30 (1) ◽  
pp. 176
Author(s):  
A. Mesalam ◽  
R. Kong ◽  
B.-H. Choi ◽  
K.-L. Lee ◽  
B.-Y. Park ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Bovine Serum ◽  
Culture Media ◽  
Fetal Bovine Serum ◽  
Developmental Competence ◽  
Mitochondrial Activity ◽  
Mrna Levels ◽  
Fetal Bovine

Serum has widely been used as a main supplement to embryo in vitro culture media as it contains embryotrophic factors. Charcoal:dextran treatment of fetal bovine serum (FBS) removes lipophilic chemicals and certain steroid hormones and growth factors. The objective of this study was to investigate the effects of charcoal:dextran-stripped fetal bovine serum (CDS FBS) and heat-inactivated FBS (HI FBS) in embryo culture medium (SOF-BE1 medium supplemented with 10% of serum) on their ability to support in vitro development of bovine embryos. The developmental ability and quality of bovine embryos were determined by assessing their cell number, lipid content, mitochondrial activity, gene expression, and cryo-tolerance. The experiment was conducted in 6 replicates (350 oocytes per group). The differences in embryo development, integrated optical intensity, and expression levels of the various genes between experimental groups were analysed by one-way ANOVA. Duncan’s multiple range tests were used to test the differences between the treatments. The level of statistical significance was set at P < 0.05. The percentages of embryos that underwent cleavage and formed a blastocyst were significantly (P < 0.05) higher in medium containing CDS FBS than in medium containing HI FBS (42.84 ± 0.78% v. 36.85 ± 0.89%, respectively). The total number of cells per Day 8 blastocyst was not significantly different (P > 0.05) between the CDS FBS group (208.40 ± 14.77) and the HI FBS group (195.11 ± 19.15). Furthermore, the beneficial effects of CDS FBS on embryos were associated with a significantly increased mitochondrial activity, as identified by MitoTracker Green, and reduced intracellular lipid content, as identified by Nile red staining, which increased their cryo-tolerance. The post-thaw survival rate of blastocysts was significantly (P < 0.05) higher after 24 h in the CDS FBS than in the HI FBS group (85.33 ± 4.84% v. 68.67 ± 1.20%). Quantitative reverse transcription PCR showed that the mRNA levels of lipid metabolism-related genes, acyl-CoA synthetase long-chain family member 3, acyl-coenzyme A dehydrogenase long-chain, and the cholesterol metabolism related gene hydroxymethylglutaryl-CoA reductase were significantly increased upon culture with CDS FBS. Moreover, the mRNA levels of survival gene sirtuin 1, antioxidant gene superoxide dismutase 2, and anti-apoptotic associated gene B-cell lymphoma 2 in frozen–thawed blastocysts were significantly (P < 0.05) higher in the CDS FBS group than in the HI FBS group; however, the mRNA level of the pro-apoptotic gene BCL2-associated X protein was significantly reduced. In conclusion, these data suggest that supplementation of in vitro culture medium with CDS FBS improves in vitro bovine embryo developmental competence and the quality of blastocysts in terms of their crytolerance and gene expression. This research was supported by grant from the Next-Generation BiogGeen21 (No. PJ01107703), IPET (No. 315017-5 and 117029-3), Allergy free cat (Co.. Felix Pets), BK21plus, and KGSP.

Tissue Engineering of Fibroblast Constructs and Anisotropic Collagen Gels

2002 ◽  
Vol 724 ◽  
Author(s):  
Sarah Calve ◽  
Ellen Arruda ◽  
Robert Dennis ◽  
Karl Grosh ◽  
Krystyna Pasyk
Keyword(s):  
Tissue Engineering ◽  
Self Assembly ◽  
Cell Layer ◽  
Fetal Bovine Serum ◽  
Self Organization ◽  
Collagen Gels ◽  
Confluent Cell ◽  
Fetal Bovine

AbstractThe creation of an in vitro functional tendon construct will enable testing of the influence of mechanics and nutrients on the development and remodeling of tendon under known controlled stimuli which is difficult to achieve in vivo. Tendon constructs were engineered in vitrovia stress-mediated self organization of fibroblasts and ECM on a laminin coated elastomer substrate. Varying the laminin density and the amount of fetal bovine serum on the substrate affected the ability of tendon fibroblasts to form a confluent cell layer and the time to layer delamination. Understanding the factors that promote self-assembly of tendon constructs will enable their combination with already developed in vitro muscle constructs.

Over Expression of MN1 Confers Resistance to Chemotherapy In Vitro and In Vivo

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 969-969
Author(s):  
Timothy Pardee ◽  
Teresa Mascenik ◽  
Britt H. Bolemon ◽  
Guerry J Cook
Keyword(s):  
Bone Marrow ◽  
The United States ◽  
Genetic Alterations ◽  
T Test ◽  
Over Expression ◽  
Student’S T ◽  
Worse Prognosis ◽  
Student’S T Test

Abstract Abstract 969 Acute myeloid leukemia (AML) is an accumulation of immature myeloid precursors that leads to progressive marrow failure and death. This disease affects approximately 12,000 people per year in the United States, causing 9,000 deaths. Despite decades of active research the overall 5 year survival remains a dismal 30–40%. The backbone of initial therapy for the last 30 years is combination chemotherapy containing cytarabine (Ara-C) and an anthracycline. Resistance to these therapies is a major problem and most patients diagnosed with AML will ultimately die from resistant disease. AML is characterized by heterogeneous genetic alterations that can be used to delineate prognosis. Using standard karyotyping techniques patients can be divided into good, intermediate and poor prognostic categories. There is a clear link between these chromosomal aberrations and response to chemotherapy as complete remission rates are significantly different between groups. Patients with no detectable cytogenetic abnormality fall into an intermediate prognostic group with a very heterogeneous outcome. Recent work has begun to uncover submicroscopic genetic alterations that effect prognosis for these patients. These alterations can be mutations, over or under expression of a particular gene. The MN1 gene encodes a transcription co-factor first identified by its involvement in a balanced translocation in a patient with a meningioma. Since its initial description it has been found over-expressed in multiple AML patient samples. There are several reports that over-expression of MN1 confers a worse prognosis in AML. High MN1 expressers were less likely to achieve a remission and had a lower 3 year survival rate. Additionally, over expression of MN1 in murine bone marrow leads to AML in transplanted recipients and predicts for resistance to ATRA in elderly AML patients. However, the effect of MN1 over expression on response to standard chemotherapy is currently unknown. To answer this question we used a murine model of AML driven by MLL-ENL. AML blasts were infected with retroviral vectors that contained MN1 and a GFP reporter. Partially infected blast populations were then exposed to various concentrations of either Ara-C or doxorubicin and the ratio of GFP positive and negative cells was compared to untreated controls. When blasts were exposed to 150 nM Ara-C the GFP+ percentage went from 21.10 (+/− 0.5302) in the control samples to 35.68 (+/−1.230) in the treated samples. This result was even more profound when cells were treated with 15 ng/ml doxorubicin where the percentage went from 21.10 (+/− 0.5302) to 80.27 (+/−1.615). Both results were highly statistically significant by two tailed student's t test with p values of 0.004 and <0.0001 respectively. Consistent results were obtained in multiple different infections and with separately derived MLL-ENL lines. These data demonstrate that blasts expressing MN1 had an advantage when exposed to either Ara-C or doxorubicin although the effect was far more pronounced with doxorubicin exposure. MN1 expressing blasts were also resistant to the combination of Ara-C and doxorubicin. In order to determine if MN1 conferred resistance to Ara-C and doxorubicin in vivo we injected sublethally irradiated, Ly5.1+ C57Bl6 recipients with a partially infected population of blasts. Ly5.1+ animals do not express the Ly5.2 allele; thus, staining cells for Ly5.2 allows differentiation of leukemic cells from endogenous marrow. Eight days after injection of blasts animals were treated with 100 mg/kg Ara-C plus 3 mg/kg doxorubicin daily for 5 days or observed. On day 6 animals were sacrificed and bone marrow from bilateral femurs was harvested, stained for Ly5.2 and analyzed by flow cytometery. Animals treated with Ara-C plus doxorubicin had 90.58% (+/−0.6638) Ly5.2+, GFP+ blasts compared to 55.38% (+/−5.245) in control animals. This result was highly statistically significant with a p value of <0.0001 by two tailed student's t test. This observation was reproducible in a separately derived MLL-ENL driven cell line. These data suggest that over expression of MN1 in this murine AML model confers resistance to both Ara-C and doxorubicin in vitro and in vivo and provides a biological explanation for the clinical observation that it confers a worse prognosis. The mechanisms involved in this resistance are currently under study. Disclosures: No relevant conflicts of interest to declare.

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