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https://www.cell.com/developmental-cell/fulltext/S1534-5807(20)30580-3

https://imgur.com/8kQMuqH

定序編碼原文節錄，中文部分請至頁底。

 Gene Editing of hESCs (BANCR Exons)
BANCRexon123_5’F_CRISPR        ATAACCATAAGTATCAACAG    BANCR exon 123 KO 5’
CRISPR
BANCRexon123_5’R_CRISPR        CTGTTGATACTTATGGTTAT    BANCR exon 123 KO 5’
CRISPR
BANCRexon123_3’F_CRISPR        AAGAAGACTTACCATGAACT    BANCR exon 123 KO 3’
CRISPR
BANCRexon123_3’R_CRISPR        AGTTCATGGTAAGTCTTCTT    BANCR exon 123 KO 3’
CRISPR
BANCRexon4_5’F_CRISPR  AGAAATGTGCACAGTAAAAC    BANCR exon 4 KO 5’ CRISPR
BANCRexon4_5’R_CRISPR  GTTTTACTGTGCACATTTCT    BANCR exon 4 KO 5’ CRISPR
BANCRexon4_3’F_CRISPR  GCTGAAACCAAAGTAGACAA    BANCR exon 4 KO 3’ CRISPR
BANCRexon4_3’R_CRISPR  TTGTCTACTTTGGTTTCAGC    BANCR exon 4 KO 3’ CRISPR
BANCR_EcoRI_exon4_F     GGGGGAATTCCTGCTGAGAAGTTCAGAGTCAA        exon4
overexpression plasmids F primer
BANCR_NotI_R    GGGGGCGGCCGCGGATCCGATTTAAATT    exon4 overexpression plasmids
R primer
CRISPR_31F      AAGAAGCCAGCCTAAACCC     Verify CRISPR 31 clones

Enhancer Knockout Region (1,026 bp)

aatagc

BANCR originated in ancestral marmosets and has subsequently been conserved
in all related primate lineages, including humans. Similar to human cells,
NHP iPSC-CMs actively express BANCR, suggesting that not only is this genomic
element conserved but so is its regulation and transcription. Surprisingly,
BANCR knockdown and over-expression affect cardiomyocyte migration, an
important phenotype in cardiac cells particularly during development.


 The predominant molecular pathway appears to be TEAD/YAP signaling, which has
gained significant interest of late, in particular in the heart where its
activity has been shown to control organ size and regeneration (Heallen et
al., 2011; Totaro et al., 2018). Although it is currently unknown whether
Hippo signaling or mechanotransduction specifically induces TEAD/YAP binding
at BANCR’s enhancer, our results do suggest that BANCR is a downstream
effector of TEAD/YAP signaling in primates, and its co-regulation by TBX5
induces its cardiac-specific expression.


In line with known TEAD/YAP signaling downstream effects, BANCR knockin
murine embryos exhibit increased heart size compared with littermate
controls. These findings raise an intriguing question regarding the potential
for a relationship between fetal cardiomyocyte migration and cardiac size,
particularly since the latter requires increased cellular proliferation that
was not detected in our murine embryo experiments.


 As discussed in the Results, this apparent discrepancy may be due to
technical issues in accurately quantifying proliferating cells in tissue
sections and may also be due to cellular proliferation occurring before the
time of embryo harvest (E16).


However, we also did not observe increased proliferation in in vitro
engineered BANCR cell lines (BKD and BOE) (Figure S2B), which argues against
a proliferative mechanism mediated directly by BANCR. In terms of heart
development, it is possible that the increased migratory potential of
BANCR-expressing fetal cardiomyocytes concomitantly stimulates cardiomyocyte
division in vivo through secondary mechanisms not seen in an artificial in
vitro environment in which cells are removed from their native context.


We do believe our findings raise the possibility of a connection between
cardiomyocyte migration and heart size that warrants follow-up investigation.
We also performed AAV BANCR gene therapy of infarcted rat hearts and found
that BANCR causes ventricular dilation.


These rat data underscore our findings that BANCR is highly expressed in
 pediatric (but not adult) dilated cardiomyopathies, a devastating disease
that results in enlargement of the heart and is the most common cause of
heart transplants in children.


BANCR may, therefore, present a therapeutic molecular target in young
patients for whom heart transplant is the only curative therapy.


 Given our findings of an
in vitro cardiomyocyte migratory phenotype that leads to an in vivo
dilatory/enlargement cardiac phenotype with BANCR expression, this study
presents a general argument for the utility of PSCs that should be
emphasized. With access to species- and disease-specific PSCs, it is now
possible to interrogate relatively pure differentiated cell populations in
vitro to yield insights into development and disease that may not make
immediate sense in the context of our historical understanding of these
processes. This is particularly true given that nearly all knowledge of human
heart development, particularly its genetic underpinnings, has been inferred
from rodent and zebrafish models. BANCR is one example of a primate-specific
genomic locus that, when studied in a context-appropriate primate cellular
model system, has yielded phenotypic results (migration) that would likely
have been missed if only rodent models were employed.


With regard to delineating a specific molecular mechanism, lncRNAs do present
significant technical challenges, particularly highly repetitive ones such as
BANCR that have endogenous retroviral origins and share sequence homology
with elements throughout the genome. These repeat sequences, which constitute
more than 70% of the BANCR transcript, prevented us from specifically and
sensitively targeting BANCR with oligonucleotides for pull-down assays that
could identify protein or DNA-binding moieties. Due to these technical
challenges, we are primarily left with indirect measures of BANCR effects on
cardiomyocytes, such as RNA-seq and ATAC-seq after knockdown, knockout, or
over-expression. These indirect measures consistently show perturbation of
pro-migratory biological pathways, such as integrin signaling, extracellular
matrix dynamics, collagen degradation, Rho GTPase signaling, etc. However,
because it is impossible to conclusively confirm a direct link between BANCR
and these pathways, we are reticent to make claims about the importance of
any one in particular. Importantly, we are confident in the TEAD/YAP
signaling pathway as a regulatory mechanism of BANCR expression, as there is
clear interaction between these factors and BANCR’s enhancer.


 The
involvement of TEAD/YAP regulation is robust and makes sense in light of the
cardiomyocyte and heart phenotypes seen with BANCR expression and knockdown.
Interestingly, integrin signaling is highly significant in the YAP1 ChIP-seq
analysis of genes near to YAP1 peaks (that include the BANCR enhancer), which
we interpret to suggest that BANCR and YAP1 overlap in regulating this
important cytoskeleton-ECM pathway in cardiomyocytes.



In broad terms, BANCR emerged after an ancient primate viral infection led to
the creation of this non-coding gene with unique, and presumably necessary,
pro-migratory function in the developing primate heart. This potent
pro-migratory effect on fetal cardiomyocytes, under regulation by TBX5 and
TEAD/YAP signaling, emphasizes the ability of ERVs to rapidly invade host
genomes and create functional elements that can alter development, disease,
and possibly primate evolution (see Graphical Abstract). It is remarkable to
consider the series of molecular and evolutionary events necessary to
accomplish this feat, especially in light of the fact that a de novo gene
(BANCR) was created that took advantage of existing cis-regulatory machinery
to specifically affect cardiomyocyte development. Starting from a single
germline insertion, the primate MER41 LTR copy-and-pasted itself to cover
approximately 0.1% of the human genome over 40–65 million years of primate
evolution. Several of these LTR copies, either stochastic or carefully
orchestrated, inserted into a favorable locus that ultimately became useful
for primate heart development. In mammalian genomes, ERVs have previously
been shown to regulate the interferon (IFN) response, a branch of innate
immunity, via generation of distributed IFN-inducible enhancers (Chuong et
al., 2016). Overall, however, functional studies of TEs in primate-specific
organismal development and evolution are lacking.



In conclusion, functional studies of primate-specific TEs such as BANCR/MER41
have been lacking due to ineffective organismal models. Human and NHP PSCs
that model development and physiology in vitro, when combined with methods
for assessing gene expression, chromatin accessibility, and conformation,
have the potential to address many of the challenges in interrogating
primate- and human-specific genomes. We expect studies such as ours will
continue to elucidate how primate retroviral elements have fundamentally
shaped our origins and our health.

內源性逆轉錄病毒衍生的一種長鏈非編碼RNA序列，被稱為BANCR。

除了出現在某些癌症中，BANCR僅僅在人類等少數幾種大型靈長動物發育中的心肌細裡
活躍。

心肌細胞是難以再生的心臟細胞，它們的協調收縮導致了心臟跳動。而BANCR特別存在於
胎兒心肌細胞中這一事實表明，它對心臟發育十分重要。



齧齒類動物體內沒有BANCR。而研究人員在胚胎小鼠中人工導入BANCR以後，動物發育出的
心臟擁有大於正常尺寸的左心室。對大鼠心臟注射一種BANCR的病毒株，在特定實驗條


「如今我們有一些證據表明，這種反轉錄病毒序列衝擊並影響了心臟的大小和功能。」
Wilson博士總結說，「我們看到，靈長類祖先身上發生了古老的病毒感染，後來所有攜帶
這些序列的靈長動物都有了更大的心臟。」


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明空覺遍大悲遊舞力 極喜任運再化利群生 直指法性水晶摩尼寶 降伏四魔無礙南瞻境
南開麗日皓月諾布尊 覺夢妙圓九乘之巔頂 善巧直趣本初普賢王 究竟證悟虹霓蓮花生
大密竅訣廣遍情器眾 本願金剛歌舞盡虛空 三門金剛無別願融入 無死住世普利界無央
https://imgur.com/Ff4PZlj 新譯白教開宗三怙主 https://imgur.com/FrXTpZe
【初音ミク】般若心経ポップ /【Hatsune Miku】 Pop Heart Sutra
https://www.youtube.com/watch?v=DCzSNssXGWI

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