335 GENETIC ENGINEERING OF GOATS FOR THE PRODUCTION OF A BIOSIMILAR ANTIBODY IN MILK

Dairy animals provide an attractive production platform for biosimilar antibodies due to the high protein production capacity of the mammary gland and easy access to milk. Goats are well suited for this approach as they offer a relatively short gestation time and good milk yield and are fully validated for the production of recombinant therapeutics. To generate transgenic goats capable of producing a biosimilar version of cetuximab, a monoclonal antibody for epidermal growth factor receptor and approved for the treatment of specific cancers, we co-transfected primary female fetal fibroblasts with expression constructs for cetuximab’s heavy (HC) and light (LC) chains under the control of the goat β-casein regulatory sequences. Beta-globin insulators were added to both transgenes to minimize position effects, and an antibiotic selection marker was placed downstream of the HC transgene sequences to allow for the isolation of stable transgenic cell clones. Selected cell clones were screened by PCR for the presence of both transgenes. Positive cell clones were analysed by Southern blot with a β-casein-specific probe. This allowed for the simultaneous detection of both transgenes, and the endogenous β-casein gene served as a standard to determine transgene copy numbers. The cell clones showed a broad range of copy numbers, from single copy insertions to >100 copies for the HC and LC transgenes. Interestingly, most of the cell clones had more LC than HC transgene copies. Ten cell clones were selected to generate transgenic founders using somatic cell nuclear transfer. We were able to produce 43 live kids from 9 cell lines following transfer of between 26 and 153 one- and two-cell embryos per line into recipients (range of 4 to 15 embryos per recipient). The one cell clone that we used unsuccessfully had the lowest number of transferred embryos (11). The efficiency for the production of live kids per transferred embryos was, on average, 5.1% (range of 1.0 to 9.7%). Kids from 5 lines were hormonally induced into lactation at the age of 10 weeks. Two lines with high copy numbers (≥30) produced either no or only a few drops of milk, whereas the lines with ≤25 transgene copies gave up to several milliliters of milk per day. Western analyses confirmed cetuximab production levels of 15 g L–1 in 2 of the lines with ≤25 transgene copies and ~45 g L–1 in a high copy number line; one low copy number line showed good HC but very low LC expression. Our data demonstrate that cetuximab can be produced in significant quantities in transgenic goats. Future work is aimed at determining production levels under natural lactation conditions and characterising glycosylation patterns to fully understand the pharmacodynamic properties of the antibody. Supported by GTC, the NZ Ministry of Science and Innovation and AgResearch.

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Developmental expression of the 412 retrotransposon in natural populations of D. melanogaster and D. simulans

Genetics Research ◽

10.1017/s0016672300004730 ◽

2000 ◽

Vol 76 (3) ◽

pp. 217-226 ◽

Cited By ~ 4

Author(s):

N. BORIE ◽

C. LOEVENBRUCK ◽

C. BIEMONT

Keyword(s):

Expression Pattern ◽

Copy Number ◽

Developmental Stages ◽

Population Sample ◽

Natural Populations ◽

Developmental Expression ◽

Regulatory Sequences ◽

Copy Numbers ◽

Species Specific ◽

Very High

We analysed the pattern of expression of retrotransposon 412 through developmental stages in various populations of Drosophila simulans and D. melanogaster differing in 412 copy number. We found that the 412 expression pattern varied greatly between populations of both species, indicating that such patterns were not entirely species-specific. In D. simulans, total transcripts increased with number of 412 copies in the chromosomes when this number was low, and then decreased for high copy numbers. D. melanogaster, which has a higher 412 copy number than D. simulans, had overall a lower global 412 expression, but again showed variation in 412 expression pattern between populations. These results suggest that in populations of D. simulans with low 412 copy number, the expression pattern of this element depends not only on copy number but also on host cellular regulatory sequences near which the elements were inserted. In D. simulans populations with high copy number overall transcription was on the contrary globally repressed, as observed in D. melanogaster. A population from Canberra (Australia) which had a very high 412 copy number was found to be associated with very high expression of 412 over all developmental stages, suggesting that the above 412 expression regulation processes are overcome in this population sample. The analysis of hybrids between geographically distinct populations of D. simulans showed that 412 expression was trans-regulated differently according to developmental stages, implying complex interactions between the 412 element and stage-specific host genes.

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Neoadjuvant Chemotherapy Induces the Appearance of New Copy Number Aberrations in Breast Tumor and is Associated with Metastasis

Current Cancer Drug Targets ◽

10.2174/1568009620666200506104523 ◽

2020 ◽

Vol 20 (9) ◽

pp. 681-688

Author(s):

Nikolai V. Litviakov ◽

Marina K. Ibragimova ◽

Matvey M. Tsyganov ◽

Artem V. Doroshenko ◽

Eugeniy Y. Garbukov ◽

...

Keyword(s):

Breast Cancer ◽

Neoadjuvant Chemotherapy ◽

Copy Number ◽

Breast Cancer Patients ◽

Free Survival ◽

Luminal B ◽

Cell Clones ◽

Genetic Landscape ◽

Intelligent Approach

Background: In this study, we examined the CNA-genetic landscape (CNA – copy number aberration) of breast cancer prior to and following neoadjuvant chemotherapy (NAC) and correlated changes in the tumor landscape with chemotherapy efficiency as well as metastasis-free survival. Objective: Breast cancer patients (n = 30) with luminal B molecular subtypes were treated with anthracycline- based therapy. Methods: To study CNAs in breast tumors, microarray analysis was performed. Results: Three effects of NAC on tumor CNA landscape were identified: 1 – the number of CNA-bearing tumor clones decreased following NAC; 2 – there were no alterations in the number of CNA-containing clones after NAC; 3 – the treatment with NAC increased the number of CNA-bearing clones (new clones appeared). All NAC-treated patients who had new tumor clones with amplification (20%) had a 100% likelihood of metastasis formation. In these cases, NAC contributed to the emergence of potential metastatic clones. Our study identified the following loci – 5p, 6p, 7q, 8q, 9p, 10p, 10q22.1, 13q, 16p, 18Chr and 19p – that were amplified during the treatment with NAC and may be the markers of potential metastatic clones. In other patients who showed total or partial elimination of CNA-bearing cell clones, no new amplification clones were observed after NAC, and no evidence of metastases was found with follow-up for 5 years (р = 0.00000). Conclusion: Our data suggest that the main therapeutic result from NAC is the elimination of potential metastatic clones present in the tumor before treatment. The results showed the necessity of an intelligent approach to NAC to avoid metastasis stimulation.

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Accucopy: accurate and fast inference of allele-specific copy number alterations from low-coverage low-purity tumor sequencing data

BMC Bioinformatics ◽

10.1186/s12859-020-03924-5 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Xinping Fan ◽

Guanghao Luo ◽

Yu S. Huang

Keyword(s):

Copy Number ◽

Bayesian Learning ◽

Kernel Smoothing ◽

Gaussian Mixture ◽

Copy Number Alterations ◽

Sequencing Data ◽

Copy Numbers ◽

Allele Specific ◽

Tumor Sequencing ◽

Low Coverage

Abstract Background Copy number alterations (CNAs), due to their large impact on the genome, have been an important contributing factor to oncogenesis and metastasis. Detecting genomic alterations from the shallow-sequencing data of a low-purity tumor sample remains a challenging task. Results We introduce Accucopy, a method to infer total copy numbers (TCNs) and allele-specific copy numbers (ASCNs) from challenging low-purity and low-coverage tumor samples. Accucopy adopts many robust statistical techniques such as kernel smoothing of coverage differentiation information to discern signals from noise and combines ideas from time-series analysis and the signal-processing field to derive a range of estimates for the period in a histogram of coverage differentiation information. Statistical learning models such as the tiered Gaussian mixture model, the expectation–maximization algorithm, and sparse Bayesian learning were customized and built into the model. Accucopy is implemented in C++ /Rust, packaged in a docker image, and supports non-human samples, more at http://www.yfish.org/software/. Conclusions We describe Accucopy, a method that can predict both TCNs and ASCNs from low-coverage low-purity tumor sequencing data. Through comparative analyses in both simulated and real-sequencing samples, we demonstrate that Accucopy is more accurate than Sclust, ABSOLUTE, and Sequenza.

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Nuclear-cytoplasmic balance: whole genome duplications induce elevated organellar genome copy number

10.1101/2021.06.08.447629 ◽

2021 ◽

Author(s):

Matheus Fernandes Gyorfy ◽

Emma R Miller ◽

Justin L Conover ◽

Corrinne E Grover ◽

Jonathan F Wendel ◽

...

Keyword(s):

Copy Number ◽

Nuclear Genome ◽

Plant Genome ◽

Whole Genome ◽

Genome Copy ◽

Relative Copy Number ◽

Organellar Genome ◽

Plastid Genomes ◽

Copy Numbers ◽

Genome Copy Number

The plant genome is partitioned across three distinct subcellular compartments: the nucleus, mitochondria, and plastids. Successful coordination of gene expression among these organellar genomes and the nuclear genome is critical for plant function and fitness. Whole genome duplication events (WGDs) in the nucleus have played a major role in the diversification of land plants and are expected to perturb the relative copy number (stoichiometry) of nuclear, mitochondrial, and plastid genomes. Thus, elucidating the mechanisms whereby plant cells respond to the cytonuclear stoichiometric imbalance that follow WGDs represents an important yet underexplored question in understanding the evolutionary consequences of genome doubling. We used droplet digital PCR (ddPCR) to investigate the relationship between nuclear and organellar genome copy numbers in allopolyploids and their diploid progenitors in both wheat and Arabidopsis. Polyploids exhibit elevated organellar genome copy numbers per cell, largely preserving the cytonuclear stoichiometry observed in diploids despite the change in nuclear genome copy number. To investigate the timescale over which cytonuclear stoichiometry may respond to WGD, we also estimated organellar genome copy number in Arabidopsis synthetic autopolyploids and in a haploid-induced diploid line. We observed corresponding changes in organellar genome copy number in these laboratory-generated lines, indicating that at least some of the cellular response to cytonuclear stoichiometric imbalance is immediate following WGD. We conclude that increases in organellar genome copy numbers represent a common response to polyploidization, suggesting that maintenance of cytonuclear stoichiometry is an important component in establishing polyploid lineages.

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Dynamics in Copy Numbers of Five Plasmids of a DairyLactococcus lactisStrain under Dairy-Related Conditions Including Near-Zero Growth Rates

Applied and Environmental Microbiology ◽

10.1128/aem.00314-18 ◽

2018 ◽

Vol 84 (11) ◽

Cited By ~ 1

Author(s):

Oscar van Mastrigt ◽

Marcel M. A. N. Lommers ◽

Yorick C. de Vries ◽

Tjakko Abee ◽

Eddy J. Smid

Keyword(s):

Nutrient Limitation ◽

Lactococcus Lactis ◽

Growth Rates ◽

Copy Number ◽

Plasmid Copy ◽

Content Type ◽

Copy Numbers ◽

Wide Range ◽

Zero Growth ◽

The Impact

ABSTRACTLactic acid bacteria can carry multiple plasmids affecting their performance in dairy fermentations. The expression of plasmid-borne genes and the activity of the corresponding proteins are severely affected by changes in the numbers of plasmid copies. We studied the impact of growth rate on the dynamics of plasmid copy numbers at high growth rates in chemostat cultures and down to near-zero growth rates in retentostat cultures. Five plasmids of the dairy strainLactococcus lactisFM03-V1 were selected, and these varied in size (3 to 39 kb), in replication mechanism (theta or rolling circle), and in putative (dairy-associated) functions. The copy numbers ranged from 1.5 to 40.5, and the copy number of theta-type replicating plasmids was negatively correlated to the plasmid size. Despite the extremely wide range of growth rates (0.0003 h−1to 0.6 h−1), the copy numbers of the five plasmids were stable and only slightly increased at near-zero growth rates, showing that the plasmid replication rate was strictly controlled. One low-copy-number plasmid, carrying a large exopolysaccharide gene cluster, was segregationally unstable during retentostat cultivations, reflected in a complete loss of the plasmid in one of the retentostat cultures. The copy number of the five plasmids was also hardly affected by varying the pH value, nutrient limitation, or the presence of citrate (maximum 2.2-fold), signifying the stability in copy number of the plasmids.IMPORTANCELactococcus lactisis extensively used in starter cultures for dairy fermentations. Important traits for the growth and survival ofL. lactisin dairy fermentations are encoded by genes located on plasmids, such as genes involved in lactose and citrate metabolism, protein degradation, oligopeptide uptake, and bacteriophage resistance. Because the number of plasmid copies could affect the expression of plasmid-borne genes, it is important to know the factors that influence the plasmid copy numbers. We monitored the plasmid copy numbers ofL. lactisat near-zero growth rates, characteristic for cheese ripening. Moreover, we analyzed the effects of pH, nutrient limitation, and the presence of citrate. This showed that the plasmid copy numbers were stable, giving insight into plasmid copy number dynamics in dairy fermentations.

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Abstract P613: Absolute Gene Quantification Of Intrarenal Renin-angiotensin System Components Using Droplet Digital PCR Analysis

Hypertension ◽

10.1161/hyp.68.suppl_1.p613 ◽

2016 ◽

Vol 68 (suppl_1) ◽

Author(s):

Ryousuke Satou ◽

Akemi Katsurada ◽

Kayoko Miyata ◽

Andrei Derbenev ◽

Andrea Zsombok

Keyword(s):

Copy Number ◽

System Analysis ◽

Renin Angiotensin System ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Gene Copy ◽

Angiotensin System ◽

Renin Angiotensin ◽

Copy Numbers ◽

Intrarenal Ras

The intrarenal renin-angiotensin system (RAS) has been shown to play crucial roles in the development of hypertension and RAS associated kidney injury including diabetic nephropathy. Although some circulating RAS components are filtered into kidneys and contribute to the regulation of intrarenal RAS activity, evaluating expression levels of RAS components in the kidney is important to elucidate the mechanisms underlying intrarenal RAS activation. Digital PCR is a new technique that has been established to quantify absolute target gene levels, which allows for comparisons of different gene levels. Thus, this study was performed to establish profiles of absolute gene copy numbers for intrarenal RAS components in wild-type (WT) rats, WT and streptozotocin (STZ)-induced diabetic mice. Male Sprague-Dawley rats (N=5) and male C57BL/6J mice were used in this study. The mice were subjected to either control (N=5) or STZ (200 mg/kg, N=4) injection. Seven days after STZ injection, copy numbers of renal cortical angiotensinogen (AGT), angiotensin-converting enzyme (ACE), ACE2, angiotensin type 1 receptor a (AT1a), and AT2 mRNA were determined by a droplet digital PCR. Since (pro)renin proteins produced by juxtaglomerular cells are secreted to circulating system, analysis of renin mRNA was excluded from this evaluation. In the renal cortex of WT rats, the copy number of AGT was higher than other measured RAS components (AGT: 719.2±46.6, ACE: 116.0±14.9, ACE2: 183.6±21.5, AT1a: 196.0±25.2 copies in 1 ng total RNA). AT2 levels were lower than other components (0.068±0.01 copies). In WT mice, ACE exhibited the highest copy number in the components (AGT: 447.2±29.0, ACE: 1662.4±61.2, ACE2: 676.8±41.5, AT1a: 867.0±16.8, AT2: 0.049±0.01 copies). Although STZ-induced diabetes did not change ACE2 and AT1a, ACE levels were reduced (765.5±98.1 copies) and AT2 levels were augmented (0.10±0.01 copies) as previously demonstrated. Accordingly, the absolute quantification by digital PCR established precise gene profiles of intrarenal RAS components, which will provide rationales for targeting the each component in future studies. Furthermore, the results indicate that the high sensitive assay accurately quantifies rare target genes including intrarenal AT2.

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Position independence and proper developmental control of gamma-globin gene expression require both a 5' locus control region and a downstream sequence element

Molecular and Cellular Biology ◽

10.1128/mcb.14.9.6087-6096.1994 ◽

1994 ◽

Vol 14 (9) ◽

pp. 6087-6096

Author(s):

Q Li ◽

J A Stamatoyannopoulos

Keyword(s):

Transgenic Mice ◽

Control Region ◽

Locus Control Region ◽

Regulatory Sequences ◽

Globin Genes ◽

Sequence Element ◽

Position Effects ◽

Developmental Control ◽

Developmental Patterns ◽

Gamma Globin

We have analyzed the expression of human gamma-globin genes during development in F2 progeny of transgenic mice carrying two types of constructs. In the first type, gamma-globin genes were linked individually to large (approximately 4-kb) sequence fragments spanning locus control region (LCR) hypersensitive site 2 (HS2) or HS3. These LCR fragments contained not only the core HS elements but also extensive evolutionarily conserved flanking sequences. The second type of construct contained tandem gamma- and beta-globin genes linked to identical HS2 or HS3 fragments. We show that gamma-globin expression in transgenic mice carrying HS2 gamma or HS3 gamma constructs is highly sensitive to position effects and that such effects override the cis regulatory elements present in these constructs to produce markedly different developmental patterns of gamma-globin expression in lines carrying the same transgene. In contrast, gamma-globin expression in both HS2 gamma beta and HS3 gamma beta mice is sheltered from position effects and the developmental patterns of gamma-globin expression in lines carrying the same transgene are identical and display stage-specific regulation. The results suggest that cis regulatory sequences required for proper developmental control of fetal globin expression in the presence of an LCR element reside downstream from the gamma genes.

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Copy Number and Prevalence of Porcine Endogenous Retroviruses (PERVs) in German Wild Boars

Viruses ◽

10.3390/v12040419 ◽

2020 ◽

Vol 12 (4) ◽

pp. 419

Author(s):

Luise Krüger ◽

Milena Stillfried ◽

Carolin Prinz ◽

Vanessa Schröder ◽

Lena Katharina Neubert ◽

...

Keyword(s):

Wild Boar ◽

Copy Number ◽

Digital Pcr ◽

Endogenous Retroviruses ◽

Wild Boars ◽

Domestic Pigs ◽

Copy Numbers ◽

Cellular Genes ◽

Porcine Endogenous Retroviruses ◽

Different Populations

Porcine endogenous retroviruses (PERVs) are integrated in the genome of pigs and are transmitted like cellular genes from parents to the offspring. Whereas PERV-A and PERV-B are present in all pigs, PERV-C was found to be in many, but not all pigs. When PERV-C is present, recombination with PERV-A may happen and the PERV-A/C recombinants are characterized by a high replication rate. Until now, nothing has been known about the copy number of PERVs in wild boars and little is known about the prevalence of the phylogenetically youngest PERV-C in ancient wild boars. Here we investigated for the first time the copy number of PERVs in different populations of wild boars in and around Berlin using droplet digital PCR. Copy numbers between 3 and 69 per genome have been measured. A lower number but a higher variability was found compared to domestic pigs, including minipigs reported earlier (Fiebig et al., Xenotransplantation, 2018). The wild boar populations differed genetically and had been isolated during the existence of the Berlin wall. Despite this, the variations in copy number were larger in a single population compared to the differences between the populations. PERV-C was found in all 92 analyzed animals. Differences in the copy number of PERV in different organs of a single wild boar indicate that PERVs are also active in wild boars, replicating and infecting new cells as has been shown in domestic pigs.

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Salivary AMY1 Copy Number Variation Modifies Age-Related Type 2 Diabetes Risk

Clinical Chemistry ◽

10.1093/clinchem/hvaa072 ◽

2020 ◽

Vol 66 (5) ◽

pp. 718-726

Author(s):

Yuwei Liu ◽

Caren E Smith ◽

Laurence D Parnell ◽

Yu-Chi Lee ◽

Ping An ◽

...

Keyword(s):

Type 2 Diabetes ◽

Copy Number Variation ◽

Metabolic Pathway ◽

Copy Number ◽

Lipid Lowering ◽

Pathway Enrichment Analysis ◽

Copy Numbers ◽

Number Variation ◽

The Relationship

Abstract Background Copy number variation (CNV) in the salivary amylase gene (AMY1) modulates salivary α-amylase levels and is associated with postprandial glycemic traits. Whether AMY1-CNV plays a role in age-mediated change in insulin resistance (IR) is uncertain. Methods We measured AMY1-CNV using duplex quantitative real-time polymerase chain reaction in two studies, the Boston Puerto Rican Health Study (BPRHS, n = 749) and the Genetics of Lipid-Lowering Drug and Diet Network study (GOLDN, n = 980), and plasma metabolomic profiles in the BPRHS. We examined the interaction between AMY1-CNV and age by assessing the relationship between age with glycemic traits and type 2 diabetes (T2D) according to high or low copy numbers of the AMY1 gene. Furthermore, we investigated associations between metabolites and interacting effects of AMY1-CNV and age on T2D risk. Results We found positive associations of IR with age among subjects with low AMY1-copy-numbers in both studies. T2D was marginally correlated with age in participants with low AMY1-copy-numbers but not with high AMY1-copy-numbers in the BPRHS. Metabolic pathway enrichment analysis identified the pentose metabolic pathway based on metabolites that were associated with both IR and the interactions between AMY1-CNV and age. Moreover, in older participants, high AMY1-copy-numbers tended to be associated with lower levels of ribonic acid, erythronic acid, and arabinonic acid, all of which were positively associated with IR. Conclusions We found evidence supporting a role of AMY1-CNV in modifying the relationship between age and IR. Individuals with low AMY1-copy-numbers tend to have increased IR with advancing age.

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Genome-Wide Identification and Analysis of High-Copy-Number LTR Retrotransposons in Asian Pears

Genes ◽

10.3390/genes10020156 ◽

2019 ◽

Vol 10 (2) ◽

pp. 156

Author(s):

Shuang Jiang ◽

Xiaoqing Wang ◽

Chunhui Shi ◽

Jun Luo

Keyword(s):

Copy Number ◽

Long Terminal Repeat Retrotransposons ◽

High Copy Number ◽

Pyrus Pyrifolia ◽

Origin And Evolution ◽

Asian Pear ◽

Copy Numbers ◽

Genome Wide ◽

History Of ◽

Insertion Sites

A large proportion of the genome of ‘Suli’ pear (Pyrus pyrifolia) contains long terminal repeat retrotransposons (LTR-RTs), which suggests that LTR-RTs have played important roles in the evolution of Pyrus. Further analysis of retrotransposons, particularly of high-copy-number LTR-RTs in different species, will provide new insights into the evolutionary history of Pyrus. A total of 4912 putative LTR-RTs classified into 198 subfamilies were identified in the ‘Suli’ pear genome. Six Asian pear accessions, including cultivars and wild species, were resequenced. The comparison of copy number for each LTR-RT subfamily was evaluated in Pyrus accessions, and data showed up to four-fold differences for some subfamilies. This contrast suggests different fates for retrotransposon families in the evolution of Pyrus. Fourteen high-copy-number subfamilies were identified in Asian pears, and more than 50% of the LTR-RTs in the genomes of all Pyrus accessions were from these 14 identified LTR-RT subfamilies. Their average insertion time was 3.42 million years ago, which suggests that these subfamilies were recently inserted into the genome. Many homologous and specific retrotransposon insertion sites were identified in oriental and occidental pears, suggesting that the duplication of retrotransposons has occurred throughout almost the entire origin and evolution of Pyrus species. The LTR-RTs show high heterogeneity, and their copy numbers vary in different Pyrus species. Thus, our findings suggest that LTR-RTs are an important source of genetic variation among Pyrus species.

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