200 Optimization of Individual Stages of Chicken Transgenesis to Increase Efficiency

2018 ◽  
Vol 30 (1) ◽  
pp. 240
Author(s):  
E. K. Tomgorova ◽  
E. N. Antonova ◽  
N. A. Volkova ◽  
P. Y. Volchkov ◽  
N. A. Zinovieva
Keyword(s):  
Cell Population ◽  
Germ Cells ◽  
Cell Sorting ◽  
Optimal Dose ◽  
Cell Types ◽  
Dorsal Aorta ◽  
Chick Embryos ◽  
Embryonic Cells ◽  
Sex Cells ◽  
Adhesive Capacity

Primordial germ cells (PGC) are the precursors of male and female progenitor cells. The cells are considered a valuable genetic material for the production of transgenic poultry. This technology includes isolation of the PGC from chick donor embryos, transformation of the cells, and injection into the dorsal aorta of recipient embryos. After injection, the PGC are involved in the process of embryo development and differentiate into male or female sex cells. The aim of the research was to optimize the individual stages of this technology to increase the efficiency of transgenesis. The PGC were extracted from embryo gonads at stage 26 to 27 (H&H) using the trypsinization process. The trypsin concentration and incubation time were determined experimentally. Treatment of chick embryos with a 0.05% trypsin solution for 5 min was optimal for obtaining culture of embryonic cells. Separation of the PGC from other types of embryonic cells was based on a differential adhesive capacity. The maximum homogeneity of the cell population for further cultivation was established by transfer (twice) of the supernatant containing unattached cells after 1 h of cultivation in a new culture dish. The cell population is represented mainly by the PGC (81 ± 4%). Additional purification of the PGC from other cell types using magnetic-activated cell sorting (MACS) increased the proportion of these cells up to 93 ± 2%. The lentiviral transduction (pHAGE vector, ZsGreen under CMV promotor) was used to transform the resulting culture of the PGC. The efficiency of infection of PGC with lentiviral particles (TU/mL = 2.5 × 108) was 70 ± 3%. The transformed cells were injected into the dorsal aorta of recipient embryos on Day 2.5 (n = 80). Before injecting donor PGC, recipient embryos were treated with busulfan to remove the endogenous PGC. The optimal dose of busulfan was selected experimentally. A series of experiments introducing busulfan in concentrations from 50 to 250 μg into chick embryos at 24 h of incubation showed that the optimal dose was 100 μg/embryo. The efficacy of colonization of gonads with donor PGC was assessed on Day-10 embryos (n = 32) and 4-week-old hatched chickens (n = 12). Cells from gonads were studied using fluorescence microscopy, fluorescence-activated cell sorting (FACS) and qPCR. The presence of fluorescent cells in the gonads of recipients was established in both embryos and hatched chickens. The relative number of the recombinant DNA copies and the relative level of expression were confirmed by qPCR. The FACS analysis of sex cells isolated from gonads of recipients showed that the percentage of transformed germ cells reached 55.8% in females (n = 5) and 31.9% in males (n = 7). Thus, the effectiveness of poultry transgenesis can be enhanced by preparation of donor PGC for injection into embryo recipients and elimination of endogenous PGC in recipients. Both the purification of PGC from other cell types based on adhesive capacity as well as treatment of embryo recipients at 24 h incubation with busulfan (100 μg/embryo) increased the effectiveness of transgenesis. Study supported by the RSF within project No. 16-16-10059.

The effect of pancreatic mesenchyme on the differentiation of endocrine cells from gastric endoderm

Development ◽  
1987 ◽  
Vol 100 (4) ◽  
pp. 661-671 ◽  
Author(s):  
B. Kramer ◽  
A. Andrew ◽  
B.B. Rawdon ◽  
P. Becker
Keyword(s):  
Endocrine Cell ◽  
Endocrine Cells ◽  
Cell Types ◽  
The Other ◽  
Chick Embryos ◽  
Cell Type ◽  
Pancreatic Insulin ◽  
Somatostatin Cells ◽  
Regional Specificity ◽  
Gut Endocrine Cells

To determine whether mesenchyme plays a part in the differentiation of gut endocrine cells, proventricular endoderm from 4- to 5-day chick or quail embryos was associated with mesenchyme from the dorsal pancreatic bud of chick embryos of the same age. The combinations were grown on the chorioallantoic membranes of host chick embryos until they reached a total incubation age of 21 days. Proventricular or pancreatic endoderm of the appropriate age and species reassociated with its own mesenchyme provided the controls. Morphogenesis in the experimental grafts corresponded closely to that in proventricular controls, i.e. the pancreatic mesenchyme supported the development of proventricular glands from proventricular endoderm. Insulin, glucagon and somatostatin cells and cells with pancreatic polypeptide-like immunoreactivity differentiated in the pancreatic controls. The latter three endocrine cell types, together with neurotensin and bombesin/gastrin-releasing polypeptide (GRP) cells, developed in proventricular controls and experimental grafts. The proportions of the major types common to proventriculus and pancreas (somatostatin and glucagon cells) were in general similar when experimental grafts were compared with proventricular controls but different when experimental and pancreatic control grafts were compared. Hence pancreatic mesenchyme did not materially affect the proportions of these three cell types in experimental grafts, induced no specific pancreatic (insulin) cell type and allowed the differentiation of the characteristic proventricular endocrine cell types, neurotensin and bombesin/GRP cells. However, an important finding was a significant reduction in the proportion of bombesin/GRP cells, attributable in part to a decrease in their number and in part to an increase in the numbers of endocrine cells of the other types. This indicates that mesenchyme may well play a part in determining the regional specificity of populations of gut endocrine cells.

Measurements of cell adhesion

Development ◽  
1973 ◽  
Vol 30 (2) ◽  
pp. 499-509
Author(s):  
Janet E. Hornby
Keyword(s):  
Cell Types ◽  
Random Motion ◽  
Chick Embryos ◽  
Collision Efficiency ◽  
20 Nm ◽  
Kidney Liver ◽  
Different Cell Types ◽  
Laminar Shear ◽  
Lyophobic Sols

Cell suspensions were prepared from the kidney, liver and heart of chick embryos of 5 or 8 days of incubation, and from the limb-buds of chick embryos of 5, 6, 7, 8 or 9 days of incubation. When these suspensions were aggregated under laminar shear in a Couette viscometer or random motion in a reciprocating shaker they obeyed the theoretical relationships derived for flocculating lyophobic sols. The values of the collision efficiency found for the different cell types under given conditions were used to calculate the force of interaction between cells of each type. The force of interaction ranged between 9 × 10−11 N (8-day heart) and 3 × 10−9 N (8-day liver). The forces of interaction between cells appear to be responsible for aligning the membranes of adjacent cells with a 10–20 nm gap. It is possible to arrange the cell types in a hierarchy based on the forces of interaction between them. The possible role of these forces in cell specificity is considered.

Simple method for isolation of primordial germ cells from chick embryos

1992 ◽  
Vol 16 (9) ◽  
pp. 853-857 ◽  
Author(s):  
I CHANG ◽  
A TAJIMA ◽  
Y YASUDA ◽  
T CHIKAMUNE ◽  
T OHNO
Keyword(s):  
Germ Cells ◽  
Primordial Germ Cells ◽  
Chick Embryos ◽  
Simple Method

scholarly journals Evolution of the mouse H-2K region: a hot spot of mutation associated with genes transcribed in embryos and/or germ cells.

Genetics ◽  
1992 ◽  
Vol 130 (3) ◽  
pp. 629-638
Author(s):  
Y I Yeom ◽  
K Abe ◽  
K Artzt
Keyword(s):  
Germ Cells ◽  
Hot Spot ◽  
Restriction Enzymes ◽  
Inbred Strains ◽  
Current View ◽  
Sequence Evolution ◽  
Embryonic Cells ◽  
Apparent Contradiction ◽  
Histocompatibility Complex ◽  
Good Vehicle

Abstract Active gene transcription is known to promote genetic change in neighboring DNA. We reasoned that the change would be readily heritable if transcription was occurring in germ cells or early embryonic cells before the germ cells are set aside. The H-2K region of the major histocompatibility complex (MHC) provides a good vehicle for testing this hypothesis because it is replete with such genes. We have compared the amount of polymorphism in 240 kb of DNA contiguous with H-2K and 150 kb of DNA flanking a homologous duplicated region in t-haplotypes and inbred strains. Using 90 probes and three restriction enzymes, we find a staggering difference in the amount of polymorphism in the H-2K region vs. the duplicated region (26% vs. 0%) of t-haplotypes. The disparity in the rate of divergence between the two regions indicates that the spatial distribution of genes and their expression pattern might be important factors in sequence evolution. Since t-haplotypes normally show extremely limited variability among themselves due to their recent divergence from a single ancestor, these results imply that the mutation rate in the H-2K region is unusually high. This is in apparent contradiction to the current view that the MHC loci have evolved at the same rate as other loci. The implications for the evolution of the H-2K gene are discussed.

scholarly journals Tissue-specific trans regulation of the mouse epigenome

2018 ◽  
Author(s):  
Christopher L. Baker ◽  
Michael Walker ◽  
Seda Arat ◽  
Guruprasad Ananda ◽  
Pavlina Petkova ◽  
...  
Keyword(s):  
Germ Cells ◽  
Es Cells ◽  
Recombinant Inbred Lines ◽  
Embryonic Stem ◽  
Cell Types ◽  
Dna Double Strand Breaks ◽  
Male Germ Cells ◽  
Tissue Specific ◽  
Natural Genetic Variation ◽  
Regulatory Loci

ABSTRACTAlthough a variety of writers, readers, and erasers of epigenetic modifications are known, we have little information about the underlying regulatory systems controlling the establishment and maintenance of the epigenetic landscape, which varies greatly among cell types. Here, we have explored how natural genetic variation impacts the epigenome in mice. Studying levels of H3K4me3, a histone modification at sites such as promoters, enhancers, and recombination hotspots, we found tissue-specific trans-regulation of H3K4me3 levels in four highly diverse cell types: male germ cells, embryonic stem (ES) cells, hepatocytes and cardiomyocytes. To identify the genetic loci involved, we measured H3K4me3 levels in male germ cells in a mapping population of 60 BXD recombinant inbred lines, identifying extensive trans-regulation primarily controlled by six major histone quantitative trait loci (hQTL). These chromatin regulatory loci act dominantly to suppress H3K4me3, which at hotspots reduces the likelihood of subsequent DNA double-strand breaks. QTL locations do not correspond with enzyme known to metabolize chromatin features. Instead their locations match clusters of zinc finger genes, making these possible candidates that explain the dominant suppression of H3K4me3. Collectively, these data describe an extensive, tissue-specific set of chromatin regulatory loci that control functionally related chromatin sites.

scholarly journals Minireview: Transcriptional Regulation of Gonadal Development and Differentiation

Endocrinology ◽  
2005 ◽  
Vol 146 (3) ◽  
pp. 1035-1042 ◽  
Author(s):  
Susan Y. Park ◽  
J. Larry Jameson
Keyword(s):  
Germ Cells ◽  
Sertoli Cells ◽  
Leydig Cells ◽  
Cell Types ◽  
Gonadal Development ◽  
Targeted Mutagenesis ◽  
Seminiferous Tubules ◽  
Molecular Pathways ◽  
Theca Cells ◽  
Main Cell

The embryonic gonad is undifferentiated in males and females until a critical stage when the sex chromosomes dictate its development as a testis or ovary. This binary developmental process provides a unique opportunity to delineate the molecular pathways that lead to distinctly different tissues. The testis comprises three main cell types: Sertoli cells, Leydig cells, and germ cells. The Sertoli cells and germ cells reside in seminiferous tubules where spermatogenesis occurs. The Leydig cells populate the interstitial compartment and produce testosterone. The ovary also comprises three main cell types: granulosa cells, theca cells, and oocytes. The oocytes are surrounded by granulosa and theca cells in follicles that grow and differentiate during characteristic reproductive cycles. In this review, we summarize the molecular pathways that regulate the distinct differentiation of these cell types in the developing testis and ovary. In particular, we focus on the transcription factors that initiate these cascades. Although most of the early insights into the sex determination pathway were based on human mutations, targeted mutagenesis in mouse models has revealed key roles for genes not anticipated to regulate gonadal development. Defining these molecular pathways provides the foundation for understanding this critical developmental event and provides new insight into the causes of gonadal dysgenesis.

Profiling Specific Inner Ear Cell Types Using Cell Sorting Techniques

2016 ◽  
pp. 431-445
Author(s):  
Taha A. Jan ◽  
Lina Jansson ◽  
Patrick J. Atkinson ◽  
Tian Wang ◽  
Alan G. Cheng
Keyword(s):  
Inner Ear ◽  
Cell Sorting ◽  
Cell Types

Avian pluripotent haemopoietic progenitor cells: detection and enrichment from the para-aortic region of the early embryo

1993 ◽  
Vol 105 (3) ◽  
pp. 661-666 ◽  
Author(s):  
F. Cormier
Keyword(s):  
Cell Population ◽  
Cell Sorting ◽  
Early Embryo ◽  
Bursa Of Fabricius ◽  
Immunoglobulin Superfamily ◽  
Avian Embryo ◽  
Erythroid Progenitors ◽  
Cell Surface Molecule ◽  
Myeloid Progenitors

In the avian embryo, the wall of the aorta is a site where haemopoiesis occurs in large diffuse foci from day 3 to day 10. In contrast to haemopoiesis in other organs of the embryo, para-aortic haemopoiesis is sustained by stem cells, which emerge in situ. Previous studies have demonstrated that the para-aortic region from the day-4 chick embryo harbours committed myeloid progenitors and committed erythroid progenitors. The present paper reports the in vitro development of para-aortic progenitors with both myelomonocytic and erythroid potentialities. Three types of myelo-erythroid progenitors were observed, giving rise to erythroblasts and monocytes, to erythroblasts and granulocytes, or to erythroblasts, monocytes and granulocytes. Their frequency in the para-aortic cell suspension was 1 per 10,000 cells. In cell sorting experiments, they co-sorted with committed progenitors in the cell population that immunolabeled with the VI-A2 monoclonal antibody, which is specific for chicken haemopoietic cells. Cell sorting also demonstrated that these multipotential progenitors did not express the BEN cell surface molecule, in contrast to late myeloid progenitors. The BEN molecule belongs to the immunoglobulin superfamily and is expressed by haemopoietic progenitors from bone marrow, selective sets of neurons and epithelial cells from the bursa of Fabricius. The myelo-erythroid progenitors were enriched 4 times in the VI-A2-positive cell population, and 2 to 5 times in the BEN-negative population. These results represent the first in vitro demonstration of avian normal myelo-erythroid progneitors.

Cellular patterning of fast and slow fibres in the intermandibularis muscle of chick embryos

Development ◽  
1993 ◽  
Vol 117 (1) ◽  
pp. 329-339 ◽  
Author(s):  
L.G. Robson
Keyword(s):  
Cell Types ◽  
Specific Pattern ◽  
Slow Muscle ◽  
Limb Bud ◽  
Muscle Fibres ◽  
Chick Embryos ◽  
Myogenic Cells ◽  
Lower Jaw ◽  
Cellular Pattern

The way in which the pattern of cell types arises during development of individual muscles was explored. The pattern of cellular differentiation resulting from the synthesis of particular fast and slow myosin heavy chains (MyHC) was investigated in the intermandibularis muscle in the lower jaw of chick embryos. The intermandibularis muscle has a proximodistal pattern of fibre type distribution. The distal region of the muscle contains a ratio of 1.5:1 fast to slow muscle fibres, which increases to > 2.5:1 in the proximal region. The intermandibularis muscle is assembled in a proximodistal sequence, with both fast and slow muscle cells differentiating within the earliest muscle and then establishing the specific pattern of cell types. This pattern is not dependent on a specific innervation source, as normal lower jaw muscles develop and the intermandibularis has the same graded cellular pattern when the mandibular primordium is grafted to the limb bud stump. Micromass cultures were used to explore the pool of potentially myogenic cells that are available to construct the muscles. Even before the muscle differentiates in vivo, both fast and slow cells are present in the primordia. These potentially myogenic cells are already distributed within the primordium in a proximodistal fashion that mimics the cellular pattern found in the muscle that develops.

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