31 Overall Goat Cloning Efficiency Under Suboptimal Conditions — A 6-Year Experience

2018 ◽  
Vol 30 (1) ◽  
pp. 155
Author(s):  
L. H. Aguiar ◽  
C. E. Méndez-Calderón ◽  
F. L. Ongaratto ◽  
R. Rossetto ◽  
D. Rondina ◽  
...  
Keyword(s):  
Well Being ◽  
Cloning Efficiency ◽  
Oocyte Competence ◽  
Animal Cloning ◽  
Chi Squared ◽  
Somatic Cell Nucleus ◽  
Technical Refinement ◽  
Over Time

Animal cloning involves a combination of several simple steps that need to be carried out at the highest efficacy to provide acceptable yet low overall cloning efficiency. Oocyte competence is key for proper somatic cell nucleus reprogramming, and technical elements must be refined to minimize interference in the overall outcome. The aim of this study was to compare the progress of a goat-cloning program over a 6-year period in which oocyte donors’ protocols and oocyte and embryo manipulation were continuously refined to cope with success under Brazilian semi-arid conditions. The cloning dataset was divided in 3 periods (P1, 2011-2012; P2, 2013-2014; P3, 2015-2016), using either in vivo- or in vitro-matured goat oocytes for cloning by nuclear transfer (NT) with different cell lines for subsequent transfer to synchronized recipients to produce transgenic liveborn kids. Over time, protocols for recovery of competent oocytes were adapted to existing conditions, also optimizing animal well being and nutritional status to attain better success. Data on total and viable oocytes, oocyte maturation, pregnancy, and cloning efficiency were analysed by the Chi-squared or t-test (P < 0.05). After 111 replicates, viable and matured oocyte rates were improved during the time, but mean number of total oocytes/donor were higher in the second period (25.4 ± 8.9 v. 18.5 ± 10.8 for P1 and 19.5 ± 6.1 for P3), whereas the mean of viable and matured oocytes were similar between P2 (19.4 ± 6.0 and 9.8 ± 4.6) and P3 (18.8 ± 7.0 and 9.6 ± 3.7), but higher than in P1 (12.8 ± 7.6 and 6.0 ± 4.1), respectively. Higher pregnancy rate was achieved in P1 and lower in P2, with both being similar to P3. However, the efficiency based on transferred embryos was similar between periods (Table 1). When compared with total oocytes, birth rate improved from the first to the last period (P1, 0.01%; P2, 0.02%; and P3, 0.09%). Cloning efficiency was measured by the number of transferred embryos compared with matured structures used for cloning, which showed an improvement over time (P1, 34.5%; P2, 43.9%; P3, 60.1%). A 5-fold enhancement was observed in the number of matured oocytes needed to produce a liveborn animal (P1, 2,779/1; P2, 1,892/1; P3, 545/1). Also, the number of donors (P1, 432; P2, 193.5; P3, 56.6) and recipients (P1, 114; P2, 57; P3, 27) needed to produce 1 animal was reduced by 7.6- and 4.2-fold between periods. In conclusion, complex and inefficient procedures such as cloning by NT require technical refinement and adjustments to existing conditions, including animal nutrition and welfare, and cumulative gain of expertise to attain successful results. Table 1.Overall goat cloning efficiency over time.

The Development of Homologous (Canine/Anti-Canine) Antibodies in Dogs with Haemophilia A (Factor VIII Deficiency): A Ten-Year Longitudinal Study

1993 ◽  
Vol 69 (01) ◽  
pp. 021-024 ◽  
Author(s):  
Shawn Tinlin ◽  
Sandra Webster ◽  
Alan R Giles
Keyword(s):  
Factor Viii ◽  
Canine Model ◽  
Significant Inhibition ◽  
Human Condition ◽  
Haemophilia A ◽  
Variable Expression ◽  
The Family ◽  
Over Time

SummaryThe development of inhibitors to factor VIII in patients with haemophilia A remains as a serious complication of replacement therapy. An apparently analogous condition has been described in a canine model of haemophilia A (Giles et al., Blood 1984; 63:451). These animals and their relatives have now been followed for 10 years. The observation that the propensity for inhibitor development was not related to the ancestral factor VIII gene has been confirmed by the demonstration of vertical transmission through three generations of the segment of the family related to a normal (non-carrier) female that was introduced for breeding purposes. Haemophilic animals unrelated to this animal have not developed functionally significant factor VIII inhibitors despite intensive factor VIII replacement. Two animals have shown occasional laboratory evidence of factor VIII inhibition but this has not been translated into clinical significant inhibition in vivo as assessed by clinical response and F.VIII recovery and survival characteristics. Substantial heterogeneity of inhibitor expression both in vitro and in vivo has been observed between animals and in individual animals over time. Spontaneous loss of inhibitors has been observed without any therapies designed to induce tolerance, etc., being instituted. There is also phenotypic evidence of polyclonality of the immune response with variable expression over time in a given animal. These observations may have relevance to the human condition both in determining the pathogenetic factors involved in this condition and in highlighting the heterogeneity of its expression which suggests the need for caution in the interpretation of the outcome of interventions designed to modulate inhibitor activity.

Abstract 5402: Two Photon Microscopy Measurements Of Sub-epicardial Sarcomere Length In Perfused Rat Hearts

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Patrizia Camelliti ◽  
Gil Bub ◽  
Daniel J Stuckey ◽  
Christian Bollensdorff ◽  
Damian J Tyler ◽  
...  
Keyword(s):  
Sarcomere Length ◽  
Left Ventricular ◽  
Model Systems ◽  
Future Research ◽  
Fundamental Parameter ◽  
Rat Hearts ◽  
Perfused Hearts ◽  
Over Time

Sarcomere length (SL) is a fundamental parameter underlying the Frank Starling relation in the heart, as it offers an absolute representation of myocardial stretch. Previous studies addressed the Frank Starling relation by measuring SL in isolated myocytes or muscle strips. Here, we report first data obtained using a novel technique to measure sub-epicardial SL in perfused hearts. Rat hearts were Langendorff perfused (normal Tyrode solution) at a constant pressure of 90mmHg, labeled with the fluorescent membrane marker di-4-ANEPPS, and then arrested with high-K + Tyrode for either 2-photon microscopy (n=4) or MRI (n=4). Image analysis software was developed to extract SL at the cell level from >1,400 2-photon images (Fig 1 ) and correct for cell angle. SL increased by 10±2 % between 30 and 80 min of perfusion (1.98±0.04 to 2.17±0.03 μm; p<0.05; Fig 1 ). Measurements of left ventricular myocardial volume (LVMV) were made in vivo and in perfused hearts using 3D MRI. LVMV increased by 24±7% from in vivo to 30 min of perfusion, and by 11±3 % between 30 and 90 min (539±35; 664±44; 737±49 mm 3 , respectively; p<0.05; Fig 1 ). We show that SL can be measured in isolated perfused hearts. The method allowed monitoring of changes in SL over time, and showed that SL and LVMV increase to a similar extent during 30–80 min perfusion with crystalloid solution, probably due to tissue oedema. This result, together with the increase in LVMV during the first 30 min, highlights the pronounced differences between in vivo , in situ , and in vitro model systems for studies of cardiac physiology and mechanics. Future research will compare changes in SL in healthy hearts and disease models involving contractile dysfunction. Figure 1: Left: 2-photon microscopy image of di-4-ANEPPS labeled myocardium. Right: SL and LVMV changes over time.

scholarly journals Time-Dependent Reduction of Calcium Oscillations in Adipose-Derived Stem Cells Differentiating towards Adipogenic and Osteogenic Lineage

Biomolecules ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 1400
Author(s):  
Enrico C. Torre ◽  
Mesude Bicer ◽  
Graeme S. Cottrell ◽  
Darius Widera ◽  
Francesco Tamagnini
Keyword(s):  
Stem Cells ◽  
Osteogenic Differentiation ◽  
Adipogenic Differentiation ◽  
Specific Treatment ◽  
Stem Cell Differentiation ◽  
Calcium Oscillations ◽  
Multipotent Stem Cells ◽  
Over Time

Adipose-derived mesenchymal stromal cells (ASCs) are multipotent stem cells which can differentiate into various cell types, including osteocytes and adipocytes. Due to their ease of harvesting, multipotency, and low tumorigenicity, they are a prime candidate for the development of novel interventional approaches in regenerative medicine. ASCs exhibit slow, spontaneous Ca2+ oscillations and the manipulation of Ca2+ signalling via electrical stimulation was proposed as a potential route for promoting their differentiation in vivo. However, the effects of differentiation-inducing treatments on spontaneous Ca2+ oscillations in ASCs are not yet fully characterised. In this study, we used 2-photon live Ca2+ imaging to assess the fraction of cells showing spontaneous oscillations and the frequency of the oscillation (measured as interpeak interval—IPI) in ASCs undergoing osteogenic or adipogenic differentiation, using undifferentiated ASCs as controls. The measurements were carried out at 7, 14, and 21 days in vitro (DIV) to assess the effect of time in culture on Ca2+ dynamics. We observed that both time and differentiation treatment are important factors associated with a reduced fraction of cells showing Ca2+ oscillations, paralleled by increased IPI times, in comparison with untreated ASCs. Both adipogenic and osteogenic differentiation resulted in a reduction in Ca2+ dynamics, such as the fraction of cells showing intracellular Ca2+ oscillations and their frequency. Adipogenic differentiation was associated with a more pronounced reduction of Ca2+ dynamics compared to cells differentiating towards the osteogenic fate. Changes in Ca2+ associated oscillations with a specific treatment had already occurred at 7 DIV. Finally, we observed a reduction in Ca2+ dynamics over time in untreated ASCs. These data suggest that adipogenic and osteogenic differentiation cell fates are associated with specific changes in spontaneous Ca2+ dynamics over time. While this observation is interesting and provides useful information to understand the functional correlates of stem cell differentiation, further studies are required to clarify the molecular and mechanistic correlates of these changes. This will allow us to better understand the causal relationship between Ca2+ dynamics and differentiation, potentially leading to the development of novel, more effective interventions for both bone regeneration and control of adipose growth.

The Impact of Two Embryo Culture Media, SOF and Commercial BO, on Pre- and Post-Implantation Development of Cloned Sannen Goat Embryos

2020 ◽  
Author(s):  
Mehdi Hajian ◽  
Farnoosh Jafarpour ◽  
Sayed Morteza Aghamiri ◽  
Shiva Rouhollahi Varnosfaderani ◽  
Mohsen Rahimi ◽  
...  
Keyword(s):  
Embryo Culture ◽  
Culture Media ◽  
Specific Gene ◽  
Limited Information ◽  
Major Drawback ◽  
Animal Cloning ◽  
The Impact ◽  
Post Implantation

Abstract Background: The ingredients of embryo culture media developed by different companies are disclosed. Thus, it is impossible to determine which ingredients might be responsible for differences in pre-and post-implantation embryo development. To address this gap, we performed an experiment to compare two embryo culture media, namely, SOF and commercial BO, on pre- and post-implantation development of cloned Sannen goat embryos. Cumulus oocyte complexes derived from slaughterhouse ovaries were used for in vitro embryo production . In vitro development of IVF, parthenogenetic and SCNT embryos were assessed in both BO and SOF media. The expression of 16 genes, including AKT , OCT4 , SOX2 , BMPR1 , FGFR4 , CDC25 , CDX2 , GCN5 , PCAF , FOXD3 , SMAD5 , FZD , LIFR1 , CTNNB , ERK1 , and IFNT , belonging to 7 important pathways, i.e. pluripotency, FGF, TGFβ, cell cycle and proliferation, histone transferase, trophectoderm, and WNT, were examined in the goat SCNT and IVF blastocysts from both BO and SOF media. Results: The blastocyst rate in BO medium was significantly higher than that of the SOF medium in SCNT embryos ( P < 0.05). All of the genes examined showed increased expression levels in SCNT embryos compared to IVF embryos. In the IVF group, OCT4 , BMPR1 , and GCN5 showed significantly higher expression in the SOF medium compared to the BO medium. In this group, AKT , FGFR4 , SOX2 showed significantly lower expression in the SOF medium compared to the BO medium. In the SCNT group, FGFR4 , GCN5 , FZD , CTNNB , BMPR1 , and FGFR4 showed significantly higher expression in SOF medium compared to BO medium. In vivo development did not differ significantly between the two groups. Conclusions: Based on these results, we concluded that the limited information available on the allocations of ICM and TE cells in SCNT embryos and embryo-specific gene expression may be the major drawback IVC medium and an impediment to successful animal cloning.

scholarly journals Intraperitoneal pressure: Stability over time and validation of Durand’s measurement method

2020 ◽  
pp. 089686082097312
Author(s):  
Alicia Sobrino-Pérez ◽  
Alfonso Pérez-Escudero ◽  
Lucila Fernández-Arroyo ◽  
Ana Dorado-García ◽  
Berta Martín-Alcón ◽  
...  
Keyword(s):  
Peritoneal Dialysis ◽  
Venous Pressure ◽  
Relevant Parameter ◽  
Pressure System ◽  
Intraperitoneal Pressure ◽  
In Vitro Measurements ◽  
Long Timescales ◽  
Over Time

Intraperitoneal pressure (IPP) is gaining consideration as a relevant parameter of peritoneal dialysis (PD) in adults, although many of its aspects are still pending clarification. We address here its stability over time and the validity of the usual method of clinical measurement, as proposed by Durand in 1992 but never specifically validated. We performed this validation by comparing Durand’s method and direct measurements with a central venous pressure system. We performed a total of 250 measurement pairs in 50 patients with different intraperitoneal volumes plus in-vitro measurements with a simulated peritoneum. Absolute differences between the two systems in vivo were 0.87 ± 0.91 cmH2O (range 0–5 cmH2O); only 6.4% of them were ≥3 cmH2O. In vitro results for both methods were identical. We also compared IPP measurements in the same patient separated by 1–4 h (514 measurement pairs in 136 patients), 1 week (92 pairs in 92 patients), and 2 years (34 pairs in 17 patients). Net differences of measurements separated by hours or 1 week were close to 0 cmH2O, with oscillations of 1.5 cmH2O in hours and 2.3 cmH2O in 1 week. IPP measured 2 years apart presented a net decrease of 2.5 ± 4.9 cmH2O, without correlation with body mass index changes or any other usual parameter of PD. In hours, 7% of IPP differences were >3 cmH2O, 22% in 1 week, and 50% in 2 years. In conclusion, Durand’s method is precise enough to measure IPP in peritoneal dialysis. This parameter is not stable over long timescales, so it is necessary to use recent measurements.

102 RABBIT CLONING: HISTONE ACETYLATION STATUS OF DONOR CELLS AND CLONED EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 168
Author(s):  
V. Zakhartchenko ◽  
F. Yang ◽  
R. Hao ◽  
E. Wolf
Keyword(s):  
Histone Acetylation ◽  
Nuclear Transfer ◽  
Epigenetic Mark ◽  
Cloning Efficiency ◽  
Developmental Potential ◽  
Acetylation Status ◽  
Donor Cells ◽  
Fetal Fibroblasts

Epigenetic status of the genome of a donor nucleus is likely to be associated with the developmental potential of cloned embryos produced by somatic cell nuclear transfer (SCNT). Prevention of epigenetic errors by manipulation of the epigenetic status of donor cells is expected to result in improvement of cloning efficiency. In this study, we transferred cultured rabbit cumulus cells (RCC) and fetal fibroblasts (RFF) from genetically marked rabbits (Ali/Bas) into metaphase II (MII) oocytes and analyzed the levels of histone H3K9 acetylation in donor cells and cloned embryos. We also assessed the correlation between the histone acetylation status of donor cells and cloned embryos and their developmental potential. To test whether alteration of the histone acetylation status affects development of cloned embryos, we treated donor cells with sodium butyrate (NaBu), a histone deacetylase inhibitor. Further, we tried to improve cloning efficiency by chimeric complementation of cloned embryos with one or two blastomeres from in vitro-fertilized or parthenogenetic embryos. Histone acetylation in donor cells and cloned embryos was detected by anti-acH3K9 antibody using Western immunoblot analysis or immunochemistry, respectively. Data were analyzed by chi-square (developmental rates) or Student-Newman-Keuls (histone acetylation) test. The levels of acetylated histone H3K9 were higher in RCCs than in RFFs (P &lt; 0.05). Although the type of donor cells did not affect development to blastocyst, after transfer into recipients, RCC-cloned embryos induced a higher initial pregnancy rate as compared to RFF-cloned embryos (40% vs. 20%; P &lt; 0.05). However, almost all pregnancies with either type of cloned embryos were lost by the middle of gestation and only one fully developed; a live RCC-derived rabbit was obtained. Treatment of RFFs with NaBu significantly (P &lt; 0.05) increased the level of histone H3K9/14 acetylation and the proportion of nuclear transfer embryos developing to blastocyst (49% vs. 33% with non-treated RFF; P &lt; 0.05). The distribution of signals for acH3K9 in either group of cloned embryos did not resemble that in in vivo-fertilized embryos, suggesting that reprogramming of this epigenetic mark is aberrant in cloned rabbit embryos and cannot be corrected by treatment of donor cells with NaBu. Aggregation of embryos cloned from NaBu-treated RFFs with blastomeres from in vivo-derived embryos improved development to blastocyst, but no cloned offspring were obtained. Two live cloned rabbits were produced from this donor cell type only after aggregation of cloned embryos with a parthenogenetic blastomere. Our study demonstrates that the levels of histone acetylation in donor cells and cloned embryos correlate with their developmental potential and can be a useful epigenetic mark to predict efficiency of SCNT rabbits. This work was supported by the Bayerische Forschungsstiftung and by Therapeutic Human Polyclonals, Inc.

239 USE OF CYCLIC ADENOSINE MONOPHOSPHATE MODULATORS IN IN VITRO PRODUCTION OF BOVINE EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 209
Author(s):  
T. Fanti ◽  
N. M. Ortega ◽  
R. Garaguso ◽  
M. J. Franco ◽  
C. Herrera ◽  
...  
Keyword(s):  
Phosphodiesterase Inhibitor ◽  
Basal Medium ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Control Group ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Oocyte Competence

In vitro embryo production systems (IVP) try to emulate and enhance molecular events that occur in in vivo reproductive systems in order to increase, not only the number of embryos generated, but also their quality. Despite advances, IVP processes are still inefficient compared with in vivo systems. Several studies have attributed this deficiency to a lack of oocyte competence due to spontaneous premature resumption of meiotic maturation in the oocyte following the removal from its follicular environment. Therefore, our objective was to increase oocyte competence avoiding premature resumption of meiosis by using cyclic adenosine monophosphate modulators. Cumulus-oocyte complexes (COC) were obtained from ovaries of slaughterhouses, washed, and randomly allocated in 2 culture systems. Oocytes in the control group (IVM) were cultured for a period of 24 h in basal medium TCM-199 with EGF (1 µg mL–1) supplemented with rhFSH (25 mIU mL–1). Oocytes in the biphasic in vitro maturation (b-IVM) group were cultured for 2 h in a basal medium supplemented with a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX, 500 µM), and an activator of adenylate cyclase (forskolin, 100 µM). Subsequently, COC were washed and cultured in basal medium supplemented with cilostamide (20 µM) and rhFSH (25 mIU mL–1) for 24 h. Maturation rates were analysed and IVF was performed with a dose of 1 × 106 sperm cells mL–1 in IVF-SOF medium. The presumptive zygotes were cultured in continuous-single-culture medium (Irvine) supplemented with 8 mg mL–1 of BSA until they reached the blastocyst stage. No significant differences in maturation, cleavage, and cryotolerance were observed between b-IVM and IVM groups (P > 0.05; Table 1). This study showed that b-IVM produced a significant increase in IVP compared with the control (IVM) at Days 7 and 8 (P < 0.01). Blastocyst hatching rate was significant (P < 0.05) for both treatment and day of analysis. The b-IVM group yielded an increase of 10 and 7.5% at Days 7 and 8, respectively, of IVP. The biphasic maturation showed an improvement in quality regarding the control group, in the timing analysis of production, and hatching percentages, and these results show that the use of cyclic adenosine monophosphate modulators in the oocyte maturation process enhances oocyte competence, which is reflected in increased productivity and embryo quality. We propose this treatment as an alternative to the standard protocols currently used in IVP of bovine embryos. Table 1.Effect of treatment on maturation, cleavage, and cryotolerance

Effect of potassium on ammoniagenesis by renal mitochondria

1976 ◽  
Vol 231 (1) ◽  
pp. 44-51 ◽  
Author(s):  
RL Tannen ◽  
AS Kunin
Keyword(s):  
Intact Animal ◽  
Absolute Magnitude ◽  
Ammonia Production ◽  
Critical Rate ◽  
Potassium Homeostasis ◽  
Effector Mechanism ◽  
Cortical Slices ◽  
Over Time

Ammonia production by rat renal cortical mitochondria was studied with both in vivo and in vitro manipulation of potassium to further elucidate the mechanisms relating potassium homeostasis and renal ammonia production. Mitochondria from potassium-depleted animals demonstrated an increase in ammonia production at all glutamine concentrations studied, which ranged from 0.5 to 10 mM. This increase in ammoniagenesis compared favorably in degree of change with and was of sufficient absolute magnitude to entirely account for the findings observed both in renal cortical slices studied in vitro and in the intact animal. When rotenone is added to the medium, increased ammoniagenesis is still detected, indicating that either glutamine entry into the mitochondria and/or the activity of phosphate-dependent glutaminase are critical rate-controlling steps. In contrast to studies with renal slices, a decrease in ammonia production was not apparent with cortical mitochondria from chronically potassium-loaded animals. In vitro alterations of the potassium homeostasis. Therefore, these experimental manipulations either do not activate an effector mechanism which takes place in vivo, or the alteration in ammoniagenesis requires an adaptation over time that is not achieved with this in vitro approach.

scholarly journals Anoikis Resistance as a Further Trait of Acidic-Adapted Melanoma Cells

2019 ◽  
Vol 2019 ◽  
pp. 1-13 ◽  
Author(s):  
Silvia Peppicelli ◽  
Jessica Ruzzolini ◽  
Francesca Bianchini ◽  
Elena Andreucci ◽  
Chiara Nediani ◽  
...  
Keyword(s):  
Blood Stream ◽  
Melanoma Cells ◽  
Extracellular Ph ◽  
Cloning Efficiency ◽  
Anoikis Resistance ◽  
Additional Mechanism ◽  
Medium Ph ◽  
Aggressive Cancer

Melanoma is characterized by a low extracellular pH, which contributes to the development of an aggressive phenotype characterized by several properties as the switch to an epithelial-to-mesenchymal program, the increase of apoptotic resistance, and the migratory ability together with the development of drug resistance. Here, we demonstrate that melanoma cells grown in low pH medium (pH 6.7) for a short (24 hours) or long (at least 3 months) period equally express an anoikis resistance profile. Anoikis is a form of apoptosis prompted by loss of adhesion, particularly requested by aggressive cancer cells to metastasize. Anoikis resistance was ascertained in acidic melanoma cells either grown in agarose-coated plates or incubated in rocking conditions. Both analyses indicate that acidic cells were more able to survive in a nonadherent condition than cells grown in standard pH, an effect resulting in a more cloning efficiency and migratory ability. Ability to survive during rocking was inhibited using mTOR/NF-kB inhibitors. Finally, we checked whether characteristics related to thein vitroanoikis resistance acquired by acidic melanoma cells might be also suitable forin vivochallenge. We injected acidic melanoma cells into blood stream, and then we verify how many cells survived in blood after 15 min from the injection. Only acidic cells, transient and chronic, survived, whereas melanoma cells grown in standard pH medium did not. Overall, we have had the opportunity to demonstrate that low extracellular pH represents an additional mechanism able to promote an anoikis resistance in solid tumors.

scholarly journals Clathrin structure characterized with monoclonal antibodies. II. Identification of in vivo forms of clathrin.

1985 ◽  
Vol 101 (6) ◽  
pp. 2055-2062 ◽  
Author(s):  
F M Brodsky
Keyword(s):  
Monoclonal Antibodies ◽  
Heavy Chain ◽  
Light Chain ◽  
Coated Vesicles ◽  
Cell Lysates ◽  
Over Time

Clathrin was isolated from detergent-solubilized, biosynthetically radiolabeled cells by immunoprecipitation with anti-clathrin monoclonal antibodies. Immunoprecipitates obtained after pulse-chase labeling demonstrated that after biosynthesis the LCa light chain of clathrin could be found either complexed to heavy chain or in a free pool (not associated with heavy chain) which decreased steadily over time. More than half of the free LCa disappeared within the first hour after biosynthesis, but some was still detectable after several hours. Incorporation of clathrin LCa light chain and heavy chain into coated vesicles was coordinate and increased up to 4 h after biosynthesis. Comparison of these kinetics suggested that once incorporated into coated vesicles, LCa and heavy chain did not dissociate, even during depolymerization of the vesicle. There was also little apparent degradation of clathrin found in coated vesicles for up to 22 h after biosynthesis. Immunoprecipitation with anti-clathrin monoclonal antibodies was carried out after fractionation of continuously radiolabeled cell lysates using two different sizing columns. These experiments indicated that the triskelion form of clathrin that has been isolated from coated vesicles in vitro also exists in vivo. They also confirmed the existence of a transient but detectable pool of newly synthesized free LCa light chain.

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