Andrei Golovko - Academia.edu (original) (raw)
Papers by Andrei Golovko
Table S4. Overall efficiency of the two-donor (2sgRNA-2ssODN) floxing method of generating the cK... more Table S4. Overall efficiency of the two-donor (2sgRNA-2ssODN) floxing method of generating the cKO alleles (XLSX 9 kb)
Table S3. The results of the two-donor floxing approach via electroporation for 7 unique loci. (X... more Table S3. The results of the two-donor floxing approach via electroporation for 7 unique loci. (XLSX 11 kb)
Table S2. The results of the two-donor floxing approach via microinjection for 49 unique loci. (X... more Table S2. The results of the two-donor floxing approach via microinjection for 49 unique loci. (XLSX 21 kb)
Table S7. The Evaluation of the modified method of two-donor floxing method (sequential delivery ... more Table S7. The Evaluation of the modified method of two-donor floxing method (sequential delivery approach) reported in the Horii et al. 2017 report. (XLSX 11 kb)
Table S6. The details and the results of alternate methods of floxing, tested on 18 loci. (XLSX 2... more Table S6. The details and the results of alternate methods of floxing, tested on 18 loci. (XLSX 23 kb)
Table S5. The details and the results of the 330 unique loci attempted for ssODNÂ knock-in projec... more Table S5. The details and the results of the 330 unique loci attempted for ssODNÂ knock-in projects (i.e. point mutation knock-in or short-tag insertion projects). (XLSX 14 kb)
Table S1. The guide RNA and singlestranded Oligonucleotide DNA sequences, their concentrations an... more Table S1. The guide RNA and singlestranded Oligonucleotide DNA sequences, their concentrations and the length of genomic regions floxed (in bp) reported in this study. (XLSX 29 kb)
Circulation, 2019
Apolipoprotein A1 (apoA1) is key to reverse cholesterol transport and HDL functionality. Global a... more Apolipoprotein A1 (apoA1) is key to reverse cholesterol transport and HDL functionality. Global apoA1 knockout alters HDL composition and impairs HDL function (JLR, 38:1033). Given that 1/3 of HDL ...
Biochemical Journal, 2006
The Gpbar1 [G-protein-coupled BA (bile acid) receptor 1] is a recently identified cell-surface re... more The Gpbar1 [G-protein-coupled BA (bile acid) receptor 1] is a recently identified cell-surface receptor that can bind and is activated by BAs, but its physiological role is unclear. Using targeted deletion of the Gpbar1 gene in mice, we show that the gene plays a critical role in the maintenance of bile lipid homoeostasis. Mice lacking Gpbar1 expression were viable, developed normally and did not show significant difference in the levels of cholesterol, BAs or any other bile constituents. However, they did not form cholesterol gallstones when fed a cholic acid-containing high-fat diet, and liver-specific gene expression indicated that Gpbar1-deficient mice have altered feedback regulation of BA synthesis. These results suggest that Gpbar1 plays a critical role in the formation of gallstones, possibly via a regulatory mechanism involving the cholesterol 7α-hydroxylase pathway.
Science, 2004
Dietary cholesterol consumption and intestinal cholesterol absorption contribute to plasma choles... more Dietary cholesterol consumption and intestinal cholesterol absorption contribute to plasma cholesterol levels, a risk factor for coronary heart disease. The molecular mechanism of sterol uptake from the lumen of the small intestine is poorly defined. We show that Niemann-Pick C1Like 1(NPC1L1) protein plays a critical role in the absorption of intestinal cholesterol. NPC1L1 expression is enriched in the small intestine and is in the brush border membrane of enterocytes. Although otherwise phenotypically normal, NPC1L1-deficient mice exhibit a substantial reduction in absorbed cholesterol, which is unaffected by dietary supplementation of bile acids. Ezetimibe, a drug that inhibits cholesterol absorption, had no effect in NPC1L1 knockout mice, suggesting that NPC1L1 resides in an ezetimibe-sensitive pathway responsible for intestinal cholesterol absorption.
Genome Biology
Background CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, p... more Background CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as “two-donor floxing” method). Results We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We ...
CRISPR–Cas9 gene editing technology has considerably facilitated the generation of mouse knockout... more CRISPR–Cas9 gene editing technology has considerably facilitated the generation of mouse knockout alleles, relieving many of the cumbersome and time-consuming steps of traditional mouse embryonic stem cell technology. However, the generation of conditional knockout alleles remains an important challenge. An earlier study reported up to 16% efficiency in generating conditional knockout alleles in mice using 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides (ssODN) (2sgRNA–2ssODN). We re-evaluated this method from a large data set generated from a consortium consisting of 17 transgenic core facilities or laboratories or programs across the world. The dataset constituted 17,887 microinjected or electroporated zygotes and 1,718 live born mice, of which only 15 (0.87%) mice harbored 2 correct LoxP insertions in cis configuration indicating a very low efficiency of the method. To determine the factors required to successfully generate conditional alleles using the 2sgRNA–...
Journal of visualized experiments : JoVE, Jun 25, 2018
Exposure to certain environmental chemicals in human and animals has been found to cause cellular... more Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic β cells which will lead to the development of type 2 diabetes mellitus (T2DM). Although the mechanisms for the chemical-induced β cell damage were unclear and likely to be complex, one recurring finding is that these chemicals induce oxidative stress leading to the generation of excessive reactive oxygen species (ROS) which induce damage to the β cell. To identify potential diabetogenic environmental chemicals, we isolated pancreatic islet cells from C57BL/6 mice and cultured islet cells in 96-well cell culture plates; then, the islet cells were dosed with chemicals and the ROS generation was detected by 2',7'-dichlorofluorescein (DCFH-DA) fluorescent dye. Using this method, we found that bisphenol A (BPA), Benzo[a]pyrene (BaP), and polychlorinated biphenyls (PCBs), could induce high levels of ROS, suggesting that they may potentially induce damage in is...
Biochemical pharmacology, Jan 22, 2018
The metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a long noncoding RNA and i... more The metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a long noncoding RNA and its overexpression is associated with the development of many types of malignancy. MALAT1 null mice show no overt phenotype. However, in transcriptome analysis of MALAT1 null mice we found significant upregulation of nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) regulated antioxidant genes including Nqo1 and Cat with significant reduction in reactive oxygen species (ROS) and greatly reduced ROS-generated protein carbonylation in hepatocyte and islets. We performed lncRNA pulldown assay using biotinylated antisense oligonucleotides against MALAT1 and found MALAT1 interacted with Nrf2, suggesting Nrf2 is transcriptionally regulated by MALAT1. Exposure to excessive ROS has been shown to cause insulin resistance through activation of c-Jun N-terminal kinase (JNK) which leads to inhibition of insulin receptor substrate 1 (IRS-1) and insulin-induced phosphorylation of serine/threonine ...
The Journal of Steroid Biochemistry and Molecular Biology
The sodium-dependent organic anion transporter SOAT (gene name SLC10A6 in man and Slc10a6 in mice... more The sodium-dependent organic anion transporter SOAT (gene name SLC10A6 in man and Slc10a6 in mice) is a plasma membrane transporter for sulfated steroids, which is highly expressed in germ cells of the testis. SOAT can transport biologically inactive sulfated steroids into specific target cells, where they can be reactivated by the steroid sulfatase (STS) to biologically active, unconjugated steroids known to regulate spermatogenesis. Significantly reduced SOAT mRNA expression was previously found in different forms of impaired spermatogenesis in man. It was supposed that SOAT plays a role for the local supply of steroids in the testis and consequently for spermatogenesis and fertility. Thus, an Slc10a6 Soat knockout mouse model was established by recombination-based target deletion of the Slc10a6 gene to elucidate the role of Soat in reproduction. However, the Slc10a6 knockout mice were fertile, produced normal litter sizes, and had normal spermatogenesis and sperm vitality. This phenotype suggests that the loss of Soat can be compensated in the knockout mice or that Soat function is not essential for reproduction. In addition to reproductive phenotyping, a comprehensive targeted steroid analysis including a set of 9 un-conjugated and 12 sulfo-conjugated steroids was performed in serum of Slc10a6 knockout and Slc10a6 wildtype mice. Only cholesterol sulfate, corticosterone, and testosterone (only in the males) could be detected in considerable amounts. Interestingly, male Slc10a6 knockout mice showed significantly higher serum levels for cholesterol sulfate compared to their wildtype controls. As cholesterol sulfate has a broader impact apart from the testis, further analysis of this phenotype will include other organs such as skin and lung, which also show high Soat expression in the mouse.
Cell Cycle
Eukaryotic initiation factor 2A (eIF2A) is a 65-kDa protein that was first identified in the earl... more Eukaryotic initiation factor 2A (eIF2A) is a 65-kDa protein that was first identified in the early 1970s as a factor capable of stimulating initiator methionyl-tRNAi (Met-tRNA Met i) binding to 40S ribosomal subunits in vitro. However, in contrast to the eIF2, which stimulates Met-tRNA Met i binding to 40S ribosomal subunits in a GTP-dependent manner, eIF2A didn't reveal any GTP-dependence, but instead was found to direct binding of the Met-tRNA Met i to 40S ribosomal subunits in a codon-dependent manner. eIF2A appears to be highly conserved across eukaryotic species, suggesting conservation of function in evolution. The yeast Saccharomyces cerevisae eIF2A null mutant revealed no apparent phenotype, however, it was found that in yeast eIF2A functions as a suppressor of internal ribosome entry site (IRES)-mediated translation. It was thus suggested that eIF2A my act by impinging on the expression of specific mRNAs. Subsequent studies in mammalian cell systems implicated eIF2A in non-canonical (non-AUG-dependent) translation initiation events involving near cognate UUG and CUG codons. Yet, the role of eIF2A in cellular functions remains largely enigmatic. As a first step toward characterization of the eIF2A function in mammalian systems in vivo, we have obtained homozygous eIF2A-total knockout (KO) mice, in which a gene trap cassette was inserted between eIF2A exons 1 and 2 disrupting expression of all exons downstream of the insertion. The KO mice strain is viable and to date displays no apparent phenotype. We believe that the eIF2A KO mice strain will serve as a valuable tool for researchers studying non-canonical initiation of translation in vivo.
Toxicology letters, Jan 17, 2017
Pregnane X receptor (PXR) plays an important role in protecting cells from mutagenic DNA damages ... more Pregnane X receptor (PXR) plays an important role in protecting cells from mutagenic DNA damages induced by endogenous and exogenous toxicants. This protective function is often attributed to the PXR-regulated metabolic detoxification. Here we report a novel potential mechanism that PXR reduces benzo-[α]-pyrene(BaP)-induced DNA damage through inhibiting the transcriptional activity of aryl hydrocarbon receptor (AhR) which plays a pivotal role in the bioactivation of BaP. We have utilized three well-characterized cell lines, i.e. Hepa1c1c7, AhR +/+; Bpr lacks AhR obligatory partner ARNT; Tao, lacks AhR, to analyze pivotal role of AhR/ARNT complex in mediating the BaP-induced DNA damages using comet assay (single-cell gel electrophoresis). We found that PXR activation could significantly inhibit BaP-induced DNA damage in the HepG2 cells as well as mouse hepatocytes. Using PXR-null and wild type mouse hepatocytes we showed that PXR activation by pregnenolone 16α-carbonitrile (PCN) sign...
Table S4. Overall efficiency of the two-donor (2sgRNA-2ssODN) floxing method of generating the cK... more Table S4. Overall efficiency of the two-donor (2sgRNA-2ssODN) floxing method of generating the cKO alleles (XLSX 9 kb)
Table S3. The results of the two-donor floxing approach via electroporation for 7 unique loci. (X... more Table S3. The results of the two-donor floxing approach via electroporation for 7 unique loci. (XLSX 11 kb)
Table S2. The results of the two-donor floxing approach via microinjection for 49 unique loci. (X... more Table S2. The results of the two-donor floxing approach via microinjection for 49 unique loci. (XLSX 21 kb)
Table S7. The Evaluation of the modified method of two-donor floxing method (sequential delivery ... more Table S7. The Evaluation of the modified method of two-donor floxing method (sequential delivery approach) reported in the Horii et al. 2017 report. (XLSX 11 kb)
Table S6. The details and the results of alternate methods of floxing, tested on 18 loci. (XLSX 2... more Table S6. The details and the results of alternate methods of floxing, tested on 18 loci. (XLSX 23 kb)
Table S5. The details and the results of the 330 unique loci attempted for ssODNÂ knock-in projec... more Table S5. The details and the results of the 330 unique loci attempted for ssODNÂ knock-in projects (i.e. point mutation knock-in or short-tag insertion projects). (XLSX 14 kb)
Table S1. The guide RNA and singlestranded Oligonucleotide DNA sequences, their concentrations an... more Table S1. The guide RNA and singlestranded Oligonucleotide DNA sequences, their concentrations and the length of genomic regions floxed (in bp) reported in this study. (XLSX 29 kb)
Circulation, 2019
Apolipoprotein A1 (apoA1) is key to reverse cholesterol transport and HDL functionality. Global a... more Apolipoprotein A1 (apoA1) is key to reverse cholesterol transport and HDL functionality. Global apoA1 knockout alters HDL composition and impairs HDL function (JLR, 38:1033). Given that 1/3 of HDL ...
Biochemical Journal, 2006
The Gpbar1 [G-protein-coupled BA (bile acid) receptor 1] is a recently identified cell-surface re... more The Gpbar1 [G-protein-coupled BA (bile acid) receptor 1] is a recently identified cell-surface receptor that can bind and is activated by BAs, but its physiological role is unclear. Using targeted deletion of the Gpbar1 gene in mice, we show that the gene plays a critical role in the maintenance of bile lipid homoeostasis. Mice lacking Gpbar1 expression were viable, developed normally and did not show significant difference in the levels of cholesterol, BAs or any other bile constituents. However, they did not form cholesterol gallstones when fed a cholic acid-containing high-fat diet, and liver-specific gene expression indicated that Gpbar1-deficient mice have altered feedback regulation of BA synthesis. These results suggest that Gpbar1 plays a critical role in the formation of gallstones, possibly via a regulatory mechanism involving the cholesterol 7α-hydroxylase pathway.
Science, 2004
Dietary cholesterol consumption and intestinal cholesterol absorption contribute to plasma choles... more Dietary cholesterol consumption and intestinal cholesterol absorption contribute to plasma cholesterol levels, a risk factor for coronary heart disease. The molecular mechanism of sterol uptake from the lumen of the small intestine is poorly defined. We show that Niemann-Pick C1Like 1(NPC1L1) protein plays a critical role in the absorption of intestinal cholesterol. NPC1L1 expression is enriched in the small intestine and is in the brush border membrane of enterocytes. Although otherwise phenotypically normal, NPC1L1-deficient mice exhibit a substantial reduction in absorbed cholesterol, which is unaffected by dietary supplementation of bile acids. Ezetimibe, a drug that inhibits cholesterol absorption, had no effect in NPC1L1 knockout mice, suggesting that NPC1L1 resides in an ezetimibe-sensitive pathway responsible for intestinal cholesterol absorption.
Genome Biology
Background CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, p... more Background CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as “two-donor floxing” method). Results We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We ...
CRISPR–Cas9 gene editing technology has considerably facilitated the generation of mouse knockout... more CRISPR–Cas9 gene editing technology has considerably facilitated the generation of mouse knockout alleles, relieving many of the cumbersome and time-consuming steps of traditional mouse embryonic stem cell technology. However, the generation of conditional knockout alleles remains an important challenge. An earlier study reported up to 16% efficiency in generating conditional knockout alleles in mice using 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides (ssODN) (2sgRNA–2ssODN). We re-evaluated this method from a large data set generated from a consortium consisting of 17 transgenic core facilities or laboratories or programs across the world. The dataset constituted 17,887 microinjected or electroporated zygotes and 1,718 live born mice, of which only 15 (0.87%) mice harbored 2 correct LoxP insertions in cis configuration indicating a very low efficiency of the method. To determine the factors required to successfully generate conditional alleles using the 2sgRNA–...
Journal of visualized experiments : JoVE, Jun 25, 2018
Exposure to certain environmental chemicals in human and animals has been found to cause cellular... more Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic β cells which will lead to the development of type 2 diabetes mellitus (T2DM). Although the mechanisms for the chemical-induced β cell damage were unclear and likely to be complex, one recurring finding is that these chemicals induce oxidative stress leading to the generation of excessive reactive oxygen species (ROS) which induce damage to the β cell. To identify potential diabetogenic environmental chemicals, we isolated pancreatic islet cells from C57BL/6 mice and cultured islet cells in 96-well cell culture plates; then, the islet cells were dosed with chemicals and the ROS generation was detected by 2',7'-dichlorofluorescein (DCFH-DA) fluorescent dye. Using this method, we found that bisphenol A (BPA), Benzo[a]pyrene (BaP), and polychlorinated biphenyls (PCBs), could induce high levels of ROS, suggesting that they may potentially induce damage in is...
Biochemical pharmacology, Jan 22, 2018
The metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a long noncoding RNA and i... more The metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a long noncoding RNA and its overexpression is associated with the development of many types of malignancy. MALAT1 null mice show no overt phenotype. However, in transcriptome analysis of MALAT1 null mice we found significant upregulation of nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) regulated antioxidant genes including Nqo1 and Cat with significant reduction in reactive oxygen species (ROS) and greatly reduced ROS-generated protein carbonylation in hepatocyte and islets. We performed lncRNA pulldown assay using biotinylated antisense oligonucleotides against MALAT1 and found MALAT1 interacted with Nrf2, suggesting Nrf2 is transcriptionally regulated by MALAT1. Exposure to excessive ROS has been shown to cause insulin resistance through activation of c-Jun N-terminal kinase (JNK) which leads to inhibition of insulin receptor substrate 1 (IRS-1) and insulin-induced phosphorylation of serine/threonine ...
The Journal of Steroid Biochemistry and Molecular Biology
The sodium-dependent organic anion transporter SOAT (gene name SLC10A6 in man and Slc10a6 in mice... more The sodium-dependent organic anion transporter SOAT (gene name SLC10A6 in man and Slc10a6 in mice) is a plasma membrane transporter for sulfated steroids, which is highly expressed in germ cells of the testis. SOAT can transport biologically inactive sulfated steroids into specific target cells, where they can be reactivated by the steroid sulfatase (STS) to biologically active, unconjugated steroids known to regulate spermatogenesis. Significantly reduced SOAT mRNA expression was previously found in different forms of impaired spermatogenesis in man. It was supposed that SOAT plays a role for the local supply of steroids in the testis and consequently for spermatogenesis and fertility. Thus, an Slc10a6 Soat knockout mouse model was established by recombination-based target deletion of the Slc10a6 gene to elucidate the role of Soat in reproduction. However, the Slc10a6 knockout mice were fertile, produced normal litter sizes, and had normal spermatogenesis and sperm vitality. This phenotype suggests that the loss of Soat can be compensated in the knockout mice or that Soat function is not essential for reproduction. In addition to reproductive phenotyping, a comprehensive targeted steroid analysis including a set of 9 un-conjugated and 12 sulfo-conjugated steroids was performed in serum of Slc10a6 knockout and Slc10a6 wildtype mice. Only cholesterol sulfate, corticosterone, and testosterone (only in the males) could be detected in considerable amounts. Interestingly, male Slc10a6 knockout mice showed significantly higher serum levels for cholesterol sulfate compared to their wildtype controls. As cholesterol sulfate has a broader impact apart from the testis, further analysis of this phenotype will include other organs such as skin and lung, which also show high Soat expression in the mouse.
Cell Cycle
Eukaryotic initiation factor 2A (eIF2A) is a 65-kDa protein that was first identified in the earl... more Eukaryotic initiation factor 2A (eIF2A) is a 65-kDa protein that was first identified in the early 1970s as a factor capable of stimulating initiator methionyl-tRNAi (Met-tRNA Met i) binding to 40S ribosomal subunits in vitro. However, in contrast to the eIF2, which stimulates Met-tRNA Met i binding to 40S ribosomal subunits in a GTP-dependent manner, eIF2A didn't reveal any GTP-dependence, but instead was found to direct binding of the Met-tRNA Met i to 40S ribosomal subunits in a codon-dependent manner. eIF2A appears to be highly conserved across eukaryotic species, suggesting conservation of function in evolution. The yeast Saccharomyces cerevisae eIF2A null mutant revealed no apparent phenotype, however, it was found that in yeast eIF2A functions as a suppressor of internal ribosome entry site (IRES)-mediated translation. It was thus suggested that eIF2A my act by impinging on the expression of specific mRNAs. Subsequent studies in mammalian cell systems implicated eIF2A in non-canonical (non-AUG-dependent) translation initiation events involving near cognate UUG and CUG codons. Yet, the role of eIF2A in cellular functions remains largely enigmatic. As a first step toward characterization of the eIF2A function in mammalian systems in vivo, we have obtained homozygous eIF2A-total knockout (KO) mice, in which a gene trap cassette was inserted between eIF2A exons 1 and 2 disrupting expression of all exons downstream of the insertion. The KO mice strain is viable and to date displays no apparent phenotype. We believe that the eIF2A KO mice strain will serve as a valuable tool for researchers studying non-canonical initiation of translation in vivo.
Toxicology letters, Jan 17, 2017
Pregnane X receptor (PXR) plays an important role in protecting cells from mutagenic DNA damages ... more Pregnane X receptor (PXR) plays an important role in protecting cells from mutagenic DNA damages induced by endogenous and exogenous toxicants. This protective function is often attributed to the PXR-regulated metabolic detoxification. Here we report a novel potential mechanism that PXR reduces benzo-[α]-pyrene(BaP)-induced DNA damage through inhibiting the transcriptional activity of aryl hydrocarbon receptor (AhR) which plays a pivotal role in the bioactivation of BaP. We have utilized three well-characterized cell lines, i.e. Hepa1c1c7, AhR +/+; Bpr lacks AhR obligatory partner ARNT; Tao, lacks AhR, to analyze pivotal role of AhR/ARNT complex in mediating the BaP-induced DNA damages using comet assay (single-cell gel electrophoresis). We found that PXR activation could significantly inhibit BaP-induced DNA damage in the HepG2 cells as well as mouse hepatocytes. Using PXR-null and wild type mouse hepatocytes we showed that PXR activation by pregnenolone 16α-carbonitrile (PCN) sign...